Pathogenesis Of T-cell Acute Lymphoblastic Leukemia Research Articles

Transcriptional regulatory program controlled by MYB in T-cell acute lymphoblastic leukemia.

The transcription factor MYB is frequently upregulated in T-cell acute lymphoblastic leukemia (T-ALL), a hematological malignancy originating from T-cell precursors. Here, we demonstrate that MYB plays a crucial role by regulating genes essential for T-ALL pathogenesis. Integrative analysis reveals a long MYB isoform, ENST00000367814.8, which is dominantly expressed and confers a proliferative advantage in T-ALL cells. Rapid depletion of MYB via dTAG-mediated protein degradation affects a large number of genes, which can be classified into early response or late response genes based on their kinetics. Early response genes include many genes involved in hematopoiesis, such as TAL1, RUNX1, GATA3, IKZF2, and CXCR4. Their expression can be recovered at later time-points, suggesting the presence of a negative feedback loop mechanism. In contrast, late response genes, which are continuously downregulated after MYB depletion, includes many genes involved in cell proliferation as well as TAL1 targets, thereby affecting the cellular phenotype.

Oncogenic dependency on SWI/SNF chromatin remodeling factors in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy arising from immature thymocytes. Unlike well-known oncogenic transcription factors, such as NOTCH1 and MYC, the involvement of chromatin remodeling factors in T-ALL pathogenesis is poorly understood. Here, we provide compelling evidence on how SWI/SNF chromatin remodeling complex contributes to human T-ALL pathogenesis. Integrative analysis of transcriptomic and ATAC-Seq datasets revealed high expression of SMARCA4, one of the subunits of the SWI/SNF complex, in T-ALL patient samples and cell lines compared to normal T cells. Loss of SMARCA protein function resulted in apoptosis induction and growth inhibition in multiple T-ALL cell lines. ATAC-Seq analysis revealed a massive reduction in chromatin accessibility across the genome after the loss of SMARCA protein function. RUNX1 interacts with SMARCA4 protein and co-occupies the same genomic regions. Importantly, the NOTCH1-MYC pathway was primarily affected when SMARCA protein function was impaired, implicating SWI/SNF as a novel therapeutic target.

Acute T-cell lymphoblastic leukemia: chimeric antigen receptor technology may offer a new hope.

Acute lymphoblastic leukemia (ALL) is a prevalent malignancy affecting the hematopoietic system, encompassing both B-cell ALL (B-ALL) and T-cell ALL (T-ALL). T-ALL, characterized by the proliferation of T-cell progenitors in the bone marrow, presents significant treatment challenges, with patients often experiencing high relapse rates and poor long-term survival despite advances in chemotherapy and hematopoietic stem cell transplantation (HSCT). This review explores the pathogenesis and traditional treatment strategies of T-ALL, emphasizing the promising potential of chimeric antigen receptor (CAR) technology in overcoming current therapeutic limitations. CAR therapy, leveraging genetically modified immune cells to target leukemia-specific antigens, offers a novel and precise approach to T-ALL treatment. The review critically analyzes recent developments in CAR-T and CAR-NK cell therapies, their common targets, optimization strategies, clinical outcomes, and the associated challenges, providing a comprehensive overview of their clinical prospects in T-ALL treatment.

Abstract A016: Novel LEF1 gene structural variants in T-cell acute lymphoblastic leukemia Patient-Derived Xenografts models

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematological disorder characterized by uncontrolled proliferation of immature T-cell precursors. LEF1 (Lymphoid Enhancer-Binding Factor 1) is a transcription factor that plays a crucial role in the development of T-cells. Recent studies have shown that LEF1 can affect the expression of MYC, a proto-oncogene that is frequently dysregulated in cancer. The highly expressed LEF1 gene and potentially its variants (K86E and P106L) stimulate cell growth by upregulating the expression of MYC. In T-ALL, MYC is often overexpressed, the abnormal expression of MYC propels the advancement of T-ALL and sustains the function of leukemia stem cells. The interaction between LEF1 and MYC in T-ALL is complex and involves multiple regulatory pathways, particularly the Wnt/β-catenin signaling pathway. Several studies have revealed a significant correlation between the inactivation of LEF1 and the overexpression of MYC in T-ALL. Therefore, understanding the role of LEF1 in T-ALL pathogenesis may provide new insights into the development of novel therapeutic strategies for this aggressive disease. This study shows that the novel structural variants (SVs) found in the LEF1 gene could be associated with the decreased expression of MYC. Methods: Leukemia cells derived from 6 patients with T-ALL were inoculated intravenously into immunodeficient mice tails. The burden of tumor was monitored within the peripheral blood (hCD45+), and the leukemia cells were collected once human CD45 was above 80%. Optical Genome Mapping (OGM, Bionano Genomics) and Whole Genome Sequencing (WGS) techniques were used to detect SVs and mutations. The findings were subsequently validated using polymerase chain reaction (PCR). Results: Of the 6 T-ALL models that were analyzed, two of them had SVs in the LEF1 gene. Although the expression of the LEF1 gene did not show significant changes, the expression of MYC was observed to be downregulated. The OGM analysis revealed that the second and third exons of the LEF1 gene, which are located in the β-catenin binding region, were deleted at the DNA level. The lack of complete identity between the sequences at the breakpoints could be attributed to DNA repair mechanisms. At the RNA level, the two models had distinct LEF1 mutations - E60K and P106L. Conclusion: In two of the six T-ALL models, novel SVs were found in the LEF1 gene that may decrease MYC expression compared to control samples. Further research is required to understand the relationship between LEF1 deletion and the regulation of MYC expression, as well as the role of this phenomenon in the development of T-ALL. Targeting LEF1 has emerged as a potential therapeutic strategy for the treatment of T-ALL. Citation Format: Yueying Wang, Wubin Qian, Mengting Qin, Xiaobo Chen, Jia Xue, Sheng Guo. Novel LEF1 gene structural variants in T-cell acute lymphoblastic leukemia Patient-Derived Xenografts models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A016.

Role of chemokines in T-cell acute lymphoblastic Leukemia: From pathogenesis to therapeutic options.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous and aggressive subtype of hematologic malignancy, with limited therapeutic options due to the complexity of its pathogenesis. Although high-dose chemotherapy and allogeneic hematopoietic stem cell transplantation have improved outcomes for T-ALL patients, there remains an urgent need for novel treatments in cases of refractory or relapsed disease. Recent research has demonstrated the potential of targeted therapies aimed at specific molecular pathways to improve patient outcomes. Chemokine-related signals, both upstream and downstream, modulate the composition of distinct tumor microenvironments, thereby regulating a multitude of intricate cellular processes such as proliferation, migration, invasion and homing. Furthermore, the progress in research has made significant contributions to precision medicine by targeting chemokine-related pathways. This review article summarizes the crucial roles of chemokines and their receptors in T-ALL pathogenesis. Moreover, it explores the advantages and disadvantages of current and potential therapeutic options that target chemokine axes, including small molecule antagonists, monoclonal antibodies, and chimeric antigen receptor T-cells.

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.

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.

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.

Identification of a Novel Curcumin Derivative Influencing Notch Pathway and DNA Damage as a Potential Therapeutic Agent in T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy considered curable by modern clinical management. Nevertheless, the prognosis for T-ALL high-risk cases or patients with relapsed and refractory disease is still dismal. Therefore, there is a keen interest in developing more efficient and less toxic therapeutic approaches. T-ALL pathogenesis is associated with Notch signaling alterations, making this pathway a highly promising target in the fight against T-ALL. Here, by exploring the anti-leukemic capacity of the natural polyphenol curcumin and its derivatives, we found that curcumin exposure impacts T-ALL cell line viability and decreases Notch signaling in a dose- and time-dependent fashion. However, our findings indicated that curcumin-mediated cell outcomes did not depend exclusively on Notch signaling inhibition, but might be mainly related to compound-induced DNA-damage-associated cell death. Furthermore, we identified a novel curcumin-based compound named CD2066, endowed with potentiated anti-proliferative activity in T-ALL compared to the parent molecule curcumin. At nanomolar concentrations, CD2066 antagonized Notch signaling, favored DNA damage, and acted synergistically with the CDK1 inhibitor Ro3306 in T-ALL cells, thus representing a promising novel candidate for developing therapeutic agents against Notch-dependent T-ALL.

Adipocyte-mediated epigenomic instability in human T-ALL cells is cytotoxic and phenocopied by epigenetic-modifying drugs.

The world’s population with obesity is reaching pandemic levels. If current trends continue, it is predicted that there will be 1.5 billion people with obesity by 2030. This projection is alarming due to the association of obesity with numerous diseases including cancer, with recent studies demonstrating a positive association with acute myeloid leukemia (AML) and B cell acute lymphoblastic leukemia (B-ALL). Interestingly, several epidemiological studies suggest the converse relationship may exist in patients with T cell acute lymphoblastic leukemia (T-ALL). To determine the relationship between obesity and T-ALL development, we employed the diet-induced obesity (DIO) murine model and cultured human T-ALL cells in adipocyte-conditioned media (ACM), bone marrow stromal cell-conditioned media, stromal conditioned media (SCM), and unconditioned media to determine the functional impact of increased adiposity on leukemia progression. Whereas only 20% of lean mice transplanted with T-ALL cells survived longer than 3 months post-inoculation, 50%–80% of obese mice with leukemia survived over this same period. Furthermore, culturing human T-ALL cells in ACM resulted in increased histone H3 acetylation (K9/K14/K18/K23/K27) and methylation (K4me3 and K27me3) posttranslational modifications (PTMs), which preceded accelerated cell cycle progression, DNA damage, and cell death. Adipocyte-mediated epigenetic changes in human T-ALL cells were recapitulated with the H3K27 demethylase inhibitor GSK-J4 and the pan-HDAC inhibitor vorinostat. These drugs were also highly cytotoxic to human T-ALL cells at low micromolar concentrations. In summary, our data support epidemiological studies demonstrating that adiposity suppresses T-ALL pathogenesis. We present data demonstrating that T-ALL cell death in adipose-rich microenvironments is induced by epigenetic modifications, which are not tolerated by leukemia cells. Similarly, GSK-J4 and vorinostat treatment induced epigenomic instability and cytotoxicity profiles that phenocopied the responses of human T-ALL cells to ACM, which provides additional support for the use of epigenetic modifying drugs as a treatment option for T-ALL.

PB1750: REARRANGEMENTS OF T-CELL RECEPTOR (TCR) LOCI IN CHILDREN WITH T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a clinically and genetically heterogeneous disease that constitutes 10%–15% of newly diagnosed pediatric ALL cases and is caused by the accumulation of genetic abnormalities that alter the mechanisms controlling normal T-cell development. The most common structural aberrations involve rearrangements of the T-cell receptor (TCR) loci – TRA/TRD (14q11) and TRB (7q34), which are found in ~25% of patients. These aberrations are the main factors initiating the events in T-ALL carcinogenesis and juxtapose TCR genes and protooncogenes that encode pivotal transcription factors, leading to their aberrant expression. The outcomes of children with T-ALL significantly improved on intensified protocols, however, ~20% of patients relapse and die owing to acquired therapy resistance. Moreover, survivors also endure the acute and long-term effects of intensive toxic chemotherapy. Aims: To analyze TCR loci rearrangements and associated genetic abnormalities in children with T-ALL treated according to BFM-based protocols, and to correlate our findings with the clinical features and the treatment responses. Methods: The bone marrow samples of 66 children diagnosed between 1996-2017 (46 boys, 20 girls, age median 7.9 years, follow-up median 86.4 months) were analyzed with cytogenomic methods. For the detection of TCR loci rearrangements and other recurrent aberrations of the genes TLX3, CDKN2A/CDKN2B and ABL1 interphase FISH (DAKO, Abbott Molecular) was used. MLPA analysis with ALL-IKZF1 probemix (MRC-Holland) was performed to detect deletions/amplifications of additional genes. Complex karyotypes were analyzed with multicolor FISH (MetaSystems) or array CGH/SNP (Agilent). Differences in OS and EFS were assessed using Kaplan-Meier method and the Mantel-Cox test. Results: Rearrangements of TCR loci were detected in 18/66 (27%) patients. Translocations involving the TRA/TRD locus were demonstrated in nine children, the TRB locus in five and the simultaneous occurrence of these aberrations in two cases, respectively. In 10/18 patients, known recurrent chromosomal translocations affecting the oncogenes TAL1 (3x), LMO2 (3x), TLX1 (2x), TAL2 (1x) and MYC (1x) were identified. In the remaining cases, TCR rearrangements were cryptic or were a part of complex karyotype. Additional chromosomal aberrations were detected in all but one patient with the deletion of CDKN2A gene being the most frequent one. Immunophenotypic data classified the patients into cortical-T (12x) and mature-T (5x) groups (1x no data). Children with aberrations of TCR loci had a significantly better prognosis - EFS (p=0.011) and OS (p=0.0074), all patients are living in the first complete remission. Summary/Conclusion: Aberrations of T-cell receptors form a genetically heterogeneous group of rearrangements, leading to aberrant expression of oncogenes involved in T-cell maturation and proliferation. They mostly occur with abnormalities of genes involved in the regulation of the cell cycle and/or signaling pathways, confirming the multistep process of T-ALL pathogenesis. In our cohort, patients with TCR rearrangements had an excellent prognosis regardless of the presence of other aberrations. Although data on larger series are clearly required, we suppose, that these children could benefit from less-intensive therapy because they may experience fewer therapeutic consequences of toxic treatment. Supported by MH CZ-DRO-VFN64165.

Arginine Methyltransferase PRMT7 Deregulates Expression of RUNX1 Target Genes in T-Cell Acute Lymphoblastic Leukemia.

Simple SummaryApproximately 15–25% of acute lymphoblastic leukemias (ALL) originate from T-lineage cells. The prognosis of T-ALL is less favorable than in B-ALL and patients experience more often relapse, which is associated with dismal survival. No established prognostic biomarkers exist for T-ALL. Here, we identified the high expression of PRMT7 in T-ALL cells. Genetic deletion of PRMT7 decreased the colony formation of T-ALL cells and altered arginine monomethylation patterns in protein complexes associated with RNA and DNA processing. Moreover, several proteins with an established role in the pathogenesis of T-ALL had disrupted arginine monomethylation patterns. Among them was RUNX1, whose target gene expression was consequently deregulated.T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.

Sirt1 Is a Novel Therapeutic Target in T-ALL

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of T-ALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstratedthe importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in T-ALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemiasleads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Identification of Novel Fusion Genes and Characterization of Transcriptome Features in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells, accounting for 15% of pediatric ALL and 25% of adult ALL. Significant therapeutic progress has been made in pediatric T-ALL, while the outcome remains poor in adult T-ALL. Therefore, it is important to further characterize the molecular pathogenesis of T-ALL. Recently, the next-generation sequencing approach allowed systematic identification of molecular markers in pediatric T-ALL.Here, by performing RNA-sequencing and other genomic analysis, we investigated genomic landscape in 130 T-ALL cases. 36 distinct gene fusions were identified, including 18 novel ones. Four types of gene abnormalities were identified among novel fusions. Of note, newly discovered ZBTB16-ABL1 and TRA-SALL2 fusions, and the involvement of NKX2-1, were recurrent events. Functional studies revealed the recurrent ZBTB16-ABL1 fusion as a leukemogenic driver. Among 48 genes with mutation rates>3%, 6 were newly found in T-ALL. Moreover, aberrant mRNA isoform of SLC17A9 gene was found in majority of cases with high expression of TAL1 . Although most fusions were mutually exclusive, they co-existed with mutations. These genetic abnormalities were correlated to deregulated gene expression markers in three transcriptome-based subgroups. The G1 group mainly harbored TLX1 or TLX3 high expression, and over-expression of HOXA gene family. The transcriptome landscape of G2 group was closely associated with NUP family genes-containing fusions. The transcriptional factors important for early hematopoiesis were also found highly expressed in this group. Notably, 91.7% of our ETP cases were clustered in G2 group. With regard to G3, all patients with STIL-TAL1 and LMO1/2-TRA were located in this group, as cases with variant fusions involving TAL1 or STIL did. The genes with transcriptional aberration related to STIL-TAL1 were found over-expressed in most cases of G3 group. Finally, we compared genomic landscapes between adult and pediatric T-ALL patients. The types of fusions seemed more diverse in children than in adults, and the mutation rates in the genes of signaling pathways and epigenetic factors, both of which been frequently seen in ETP-ALL, were much higher in adults than in children. Our data suggest that the two T-ALL age groups might represent distinct disease entities, which could explain, at least in part, their distinct clinical outcomes. This study shed new lights on the leukemogenesis of T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Aging of preleukemic thymocytes drives CpG island hypermethylation in T-cell acute lymphoblastic leukemia.

Cancer cells display DNA hypermethylation at specific CpG islands in comparison to their normal healthy counterparts, but the mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. Here, we show that CpG island methylation in human T-cell acute lymphoblastic leukemia (T-ALL) mainly occurs at promoters of Polycomb Repressor Complex 2 (PRC2) target genes that are not expressed in normal or malignant T-cells and which display a reciprocal association with H3K27me3 binding. In addition, we revealed that this aberrant methylation profile reflects the epigenetic history of T-ALL and is established already in pre-leukemic, self-renewing thymocytes that precede T-ALL development. Finally, we unexpectedly uncover that this age-related CpG island hypermethylation signature in T-ALL is completely resistant to the FDA-approved hypomethylating agent Decitabine. Altogether, we here provide conceptual evidence for the involvement of a pre-leukemic phase characterized by self-renewing thymocytes in the pathogenesis of human T-ALL.

LncRNA CDKN2B-AS1 contributes to tumorigenesis and chemoresistance in pediatric T-cell acute lymphoblastic leukemia through miR-335-3p/TRAF5 axis.

T-cell acute lymphoblastic leukemia (T-ALL) is the most prevalent malignancy in children. Long non-coding RNAs are being found to have relevance to the pathogenesis of pediatric T-ALL. However, the function of cyclin-dependent kinase inhibitor 2B anti-sense RNA 1 (CDKN2B-AS1) in pediatric T-ALL progression and chemoresistance has not been illuminated. The levels of CDKN2B-AS1, miR-335-3p and tumor necrosis factor receptor-associated factor 5 (TRAF5) were assessed by quantitative real-time PCR. Cell proliferation and the 50% inhibitory concentration (IC50) value were detected using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetr-azolium assay. Cell cycle and apoptosis were evaluated by flow cytometry. Western blot analysis was performed to measure protein expression. Targeted interactions among CDKN2B-AS1, miR-335-3p and TRAF5 were determined by the dual-luciferase reporter and RNA immunoprecipitation assays. Animal studies were conducted to observe the function of CDKN2B-AS1 in vivo. Our data indicated that CDKN2B-AS1 was highly expressed in pediatric T-ALL peripheral blood mononuclear cells and cells, and high CDKN2B-AS1 level was associated with adriamycin (ADR) resistance. CDKN2B-AS1 depletion hindered T-ALL/ADR cell proliferation and cell cycle progression, and promoted cell apoptosis and ADR sensitivity in vitro. Moreover, CDKN2B-AS1 knockdown repressed tumor growth and enhanced ADR sensitivity in vivo. CDKN2B-AS1 sequestered miR-335-3p, and CDKN2B-AS1 depletion exerted regulatory effect in T-ALL/ADR cell progression by up-regulating miR-335-3p. TRAF5 was a direct target of miR-335-3p, and TRAF5 mediated the regulation of miR-335-3p in T-ALL cell behaviors. Furthermore, CDKN2B-AS1 positively modulated TRAF5 expression through sponging miR-335-3p. The current work suggested that CDKN2B-AS1 knockdown repressed the progression and enhanced ADR sensitivity of pediatric T-ALL at least partly through targeting miR-335-3p/TRAF5 axis, highlighting a promising therapeutic target for the treatment of pediatric T-ALL patients.

Abstract LB-301: Oncorequisite role of an aldehyde dehydrogenase in the pathogenesis of T-cell acute lymphoblastic leukemia

Abstract Aldehyde dehydrogenases (ALDHs) have been implicated as a stem cell marker both in normal and malignant cells. Elevated ALDH activity is associated with the stemness capability as well as the drug resistance in various cancers. As a notable example, ALDH1A2, one of ALDH family genes, is ectopically overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological disease that results from malignant transformation of T-cell progenitors. However, its roles and molecular functions in T-ALL pathogenesis are poorly understood. In our study, we demonstrated that the oncogenic transcription factor TAL1 directly induces an expression of ALDH1A2 in T-ALL cells, while normal T-cells do not express this gene. The TAL1 transcriptional complex binds to an intragenic regulatory element of ALDH1A2 and aberrantly activates the alternative promoter of the short isoform, which is specific to T-ALL cells. Using in vitro enzymatic assay, we found that the short isoform still retain enzymatic activity in catalyzing the oxidation of aldehydes. Importantly, ALDH1A2 promotes the viability and survival of T-ALL cells. Unbiased gene expression and metabolome profiling demonstrated that ALDH1A2 promotes glycolysis and mitochondria respiration, thereby supporting energy production. Additionally, expression of the short ALDH1A2 attenuated the amount of reactive oxygen species (ROS) both in vitro and in vivo. Furthermore, forced expression of short ALDH1A2 in T-cells increased overall penetrance of T-cell malignancy induced by the AKT2 oncogene in a zebrafish model. Taken together, our data suggests that ALDH1A2 is crucial and advantageous for T-ALL cell viability and survival by mediating energy production and by protecting cancer cells from oxidative stresses, thus serving as a requisite to maintain the hyperproliferative state of T-ALL cells (“onco-requisite”). Citation Format: CHUJING ZHANG, Stella Amanda, Cheng Wang, Tze King Tan, Zulfaqar Ali Muhammad, Wei Zhong Leong, Ley Moy Ng, Shojiro Kitajima, Zhenhua Li, Allen Eng Juh Yeoh, Shi Hao Tan, Takaomi Sanda. Oncorequisite role of an aldehyde dehydrogenase in the pathogenesis of T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-301.

Oncorequisite role of an aldehyde dehydrogenase in the pathogenesis of T-cell acute lymphoblastic leukemia

Aldehyde dehydrogenases (ALDH) are overexpressed in various types of cancers. One of the ALDH family genes, ALDH1A2, is aberrantly expressed in more than 50% of cases of T-cell acute lymphoblastic leukemia (T-ALL). However, its molecular function and role in the pathogenesis of T-ALL are largely unknown. Chromatin immunoprecipitation-sequencing and RNA-sequencing analyses showed that the oncogenic transcription factor TAL1 and its regulatory partners bind to the intronic regulatory element of the ALDH1A2 gene, directly inducing a T-ALL-specific isoform with enzymatic activity. ALDH1A2 was preferentially expressed in the TAL1-positive T-ALL subgroup. In TALL cell lines, depletion of ALDH1A2 inhibited cell viability and induced apoptosis. Interestingly, gene expression and metabolomic profiling revealed that ALDH1A2 supported glycolysis and the tricarboxylic acid cycle, accompanied by NADH production, by affecting multiple metabolic enzymes to promote ATP production. Depletion of ALDH1A2 increased the levels of reactive oxygen species, while the levels were reduced by ALDH1A2 overexpression both in vitro and in vivo. Overexpression of ALDH1A2 accelerated tumor onset and increased tumor penetrance in a zebrafish model of T-ALL. Taken together, our results indicate that ALDH1A2 protects against intracellular stress and promotes T-ALL cell metabolism and survival. ALDH1A2 overexpression enables leukemic clones to sustain a hyper-proliferative state driven by oncogenes.

MiR-29b-3p cooperates with miR-29c-3p to affect the malignant biological behaviors in T-cell acute lymphoblastic leukemia via TFAP2C/GPX1 axis

Mounting evidence has illustrated the tumor regulatory roles of microRNAs (miRNAs) in T-cell acute lymphoblastic leukemia (T-ALL), a malignant carcinoma originated from T-cell precursors. However, the possible regulation mechanisms underlying miR-29b/29c-3p in T-ALL have not been interrogated yet. The aim of our study was to probe the association and possible molecular mechanism of miR-29b/29c-3p and Glutathione Peroxidase 1 (GPX1), a predicted highly expressed gene in acute myeloid leukemia (LAML) tissues on the cancer genome atlas (TCGA) website. In our paper, it was observed that GPX1 was relatively overexpressed in T-ALL cells, compared with normal T cells. Loss-of-function assays demonstrated that GPX1 knockdown inhibited the proliferation and activated the apoptosis in T-ALL cells. Then miR-29b/29c-3p was confirmed to regulate GPX1 mRNA and protein expression via decreasing Transcription Factor AP-2 Gamma (TFAP2C) expression. In summary, miR-29b-3p and miR-29c-3p targeted TFAP2C so as to repress GPX1 transcription, thereafter inhibiting GPXA expression. In the end, rescue experiments proved the whole regulation mechanism of miR-29b/29c-3p in T-ALL. Overall, the miR-29b/29c-3p -TFAP2C-GPX1 axis helped us to have a better understanding of T-ALL pathogenesis.

NF-κB1 Regulates Immune Environment and Outcome of Notch-Dependent T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric malignancy that arises from the transformation of immature T-cell progenitors and has no definitive cure. Notch signaling governs many steps of T cell development and its dysregulation represents the most common causative event in the pathogenesis of T-ALL. The activation of canonical NF-κB pathway has been described as a critical downstream mediator of Notch oncogenic functions, through the sustaining of tumor cell survival and growth. The potential role of Notch/NF-κB partnership is also emerging in the generation and function of regulatory T cells (Tregs) in the context of cancer. However, little is known about the effects of combined mutations of Notch and NF-κB in regulating immune-environment and progression of T-ALL. To shed light on the topics above we generated double-mutant mice, harboring conventional knock-out mutation of NF-κB1/p50 on the genetic background of a transgenic model of Notch-dependent T-ALL. The immunophenotyping of double-mutant mice demonstrates that NF-κB1 deletion inhibits the progression of T-ALL and strongly modifies immune-environment of the disease. Double-mutant mice display indeed a dramatic reduction of pre-leukemic CD4+CD8+ (DP) T cells and regulatory T cells (Tregs) and, concurrently, the rising of an aggressive myeloproliferative trait with a massive expansion of CD11b+Gr-1+ cells in the periphery, and an accumulation of the granulocyte/monocyte progenitors in the bone-marrow. Interestingly, double-mutant T cells are able to improve the growth of CD11b+Gr-1+ cells in vitro, and, more importantly, the in vivo depletion of T cells in double-mutant mice significantly reduces the expansion of myeloid compartment. Our results strongly suggest that the myeloproliferative trait observed in double-mutant mice may depend on non-cell-autonomous mechanism/s driven by T cells. Moreover, we demonstrate that the reduction of CD4+CD8+ (DP) T cells and Tregs in double-mutant mice relies on a significant enhancement of their apoptotic rate. In conclusion, double-mutant mice may represent a useful model to deepen the knowledge of the consequences on T-ALL immune-environment of modulating Notch/NF-κB relationships in tumor cells. More importantly, information derived from these studies may help in the refinement of multitarget therapies for the disease.