How to resist Notch-targeted T-leukemia therapy: Lineage- and MYC enhancer switch.

Gain-of-function NOTCH1 mutations drive oncogenic MYC expression in T-ALL cells. Zhou et al. (2022) reveal that Notch-targeted therapy-resistant T-ALL cells activate EBF1, which promotes a T-to-B lineage shift and maintains oncogenic MYC expression in the absence of Notch signaling.

Genome-wide mapping and characterization of Notch-regulated long noncoding RNAs in acute leukemia.

Notch signaling is a key developmental pathway that is subject to frequent genetic and epigenetic perturbations in many different human tumors. Here we investigate whether long noncoding RNA (lncRNA) genes, in addition to mRNAs, are key downstream targets of oncogenic Notch1 in human T cell acute lymphoblastic leukemia (T-ALL). By integrating transcriptome profiles with chromatin state maps, we have uncovered many previously unreported T-ALL-specific lncRNA genes, a fraction of which are directly controlled by the Notch1/Rpbjκ activator complex. Finally we have shown that one specific Notch-regulated lncRNA, LUNAR1, is required for efficient T-ALL growth in vitro and in vivo due to its ability to enhance IGF1R mRNA expression and sustain IGF1 signaling. These results confirm that lncRNAs are important downstream targets of the Notch signaling pathway, and additionally they are key regulators of the oncogenic state in T-ALL.

The ubiquitin ligase FBXW7 modulates leukemia-initiating cell activity by regulating MYC stability.

Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.

Valine tRNA levels and availability regulate complex I assembly in leukaemia.

Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers1,2, the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.

Clonal evolution dissection reveals that a high MSI2 level promotes chemoresistance in T-cell acute lymphoblastic leukemia.

ABSTRACT: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer with resistant clonal propagation in recurrence. We performed high-throughput droplet-based 5' single-cell RNA with paired T-cell receptor (TCR) sequencing of paired diagnosis-relapse (Dx_Rel) T-ALL samples to dissect the clonal diversities. Two leukemic evolutionary patterns, "clonal shift" and "clonal drift" were unveiled. Targeted single-cell DNA sequencing of paired Dx_Rel T-ALL samples further corroborated the existence of the 2 contrasting clonal evolution patterns, revealing that dynamic transcriptional variation might cause the mutationally static clones to evolve chemotherapy resistance. Analysis of commonly enriched drifted gene signatures showed expression of the RNA-binding protein MSI2 was significantly upregulated in the persistent TCR clonotypes at relapse. Integrated in vitro and in vivo functional studies suggested that MSI2 contributed to the proliferation of T-ALL and promoted chemotherapy resistance through the posttranscriptional regulation of MYC, pinpointing MSI2 as an informative biomarker and novel therapeutic target in T-ALL.

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.

Endothelial cell-leukemia interactions remodel drug responses, uncovering T-ALL vulnerabilities.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and often incurable disease. To uncover therapeutic vulnerabilities, we first developed T-ALL patient-derived tumor xenografts (PDXs) and exposed PDX cells to a library of 433 clinical-stage compounds in vitro. We identified 39 broadly active drugs with antileukemia activity. Because endothelial cells (ECs) can alter drug responses in T-ALL, we developed an EC/T-ALL coculture system. We found that ECs provide protumorigenic signals and mitigate drug responses in T-ALL PDXs. Whereas ECs broadly rescued several compounds in most models, for some drugs the rescue was restricted to individual PDXs, suggesting unique crosstalk interactions and/or intrinsic tumor features. Mechanistically, cocultured T-ALL cells and ECs underwent bidirectional transcriptomic changes at the single-cell level, highlighting distinct "education signatures." These changes were linked to bidirectional regulation of multiple pathways in T-ALL cells as well as in ECs. Remarkably, in vitro EC-educated T-ALL cells transcriptionally mirrored ex vivo splenic T-ALL at single-cell resolution. Last, 5 effective drugs from the 2 drug screenings were tested in vivo and shown to effectively delay tumor growth and dissemination thus prolonging overall survival. In sum, we developed a T-ALL/EC platform that elucidated leukemia-microenvironment interactions and identified effective compounds and therapeutic vulnerabilities.

IL-7 receptor expression is frequent in T-cell acute lymphoblastic leukemia and predicts sensitivity to JAK inhibition.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis related to refractory/relapsing diseases, raising the need for new targeted therapies. Activating mutations of interleukin-7-receptor pathway genes (IL-7Rp) play a proven leukemia-supportive role in T-ALL. JAK inhibitors, such as ruxolitinib, have recently demonstrated preclinical efficacy. However, prediction markers for sensitivity to JAK inhibitors are still lacking. Herein, we show that IL-7R (CD127) expression is more frequent (∼70%) than IL-7Rp mutations in T-ALL (∼30%). We compared the so-called nonexpressers (no IL-7R expression/IL-7Rp mutation), expressers (IL7R expression without IL-7Rp mutation), and mutants (IL-7Rp mutations). Integrative multiomics analysis outlined IL-7R deregulation in virtually all T-ALL subtypes, at the epigenetic level in nonexpressers, genetic level in mutants, and posttranscriptional level in expressers. Ex vivo data using primary-derived xenografts support that IL-7Rp is functional whenever the IL-7R is expressed, regardless of the IL-7Rp mutational status. Consequently, ruxolitinib impaired T-ALL survival in both expressers and mutants. Interestingly, we show that expressers displayed ectopic IL-7R expression and IL-7Rp addiction conferring a deeper sensitivity to ruxolitinib. Conversely, mutants were more sensitive to venetoclax than expressers. Overall, the combination of ruxolitinib and venetoclax resulted in synergistic effects in both groups. We illustrate the clinical relevance of this association by reporting the achievement of complete remission in 2 patients with refractory/relapsed T-ALL. This provides proof of concept for translation of this strategy into clinics as a bridge-to-transplantation therapy. IL7R expression can be used as a biomarker for sensitivity to JAK inhibition, thereby expanding the fraction of patients with T-ALL eligible for ruxolitinib up to nearly ∼70% of T-ALL cases.

STAT5 activation promotes progression and chemotherapy resistance in early T-cell precursor acute lymphoblastic leukemia.

Interleukin-7 (IL-7) supports the growth and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL), particularly the early T-cell precursor subtype (ETP-ALL), which frequently has activating mutations of IL-7 signaling. Signal transducer and activator of transcription (STAT5) is an attractive therapeutic target because it is almost universally activated in ETP-ALL, even in the absence of mutations of upstream activators such as the IL-7 receptor (IL-7R), Janus kinase, and Fms-like tyrosine kinase 3 (FLT3). To examine the role of activated STAT5 in ETP-ALL, we have used a Lmo2-transgenic (Lmo2Tg) mouse model in which we can monitor chemoresistant preleukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs) that drive T-ALL development and relapse following chemotherapy. Using IL-7R-deficient Lmo2Tg mice, we show that IL-7 signaling was not required for the formation of pre-LSCs but essential for their expansion and clonal evolution into LSCs to generate T-ALL. Activated STAT5B was sufficient for the development of T-ALL in IL-7R-deficient Lmo2Tg mice, indicating that inhibition of STAT5 is required to block the supportive signals provided by IL-7. To further understand the role of activated STAT5 in LSCs of ETP-ALL, we developed a new transgenic mouse that enables T-cell specific and doxycycline-inducible expression of the constitutively activated STAT5B1∗6 mutant. Expression of STAT5B1∗6 in T cells had no effect alone but promoted expansion and chemoresistance of LSCs in Lmo2Tg mice. Pharmacologic inhibition of STAT5 with pimozide-induced differentiation and loss of LSCs, while enhancing response to chemotherapy. Furthermore, pimozide significantly reduced leukemia burden in vivo and overcame chemoresistance of patient-derived ETP-ALL xenografts. Overall, our results demonstrate that STAT5 is an attractive therapeutic target for eradicating LSCs in ETP-ALL.

Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress.

ABSTRACT: Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL), but pan-Notch inhibitors showed excessive toxicity in clinical trials. To find alternative ways to target Notch signals, we investigated cell division cycle 73 (Cdc73), which is a Notch cofactor and key component of the RNA polymerase-associated transcriptional machinery, an emerging target in T-ALL. Although we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, chromatin and nascent gene expression profiling showed that Cdc73 intersects with Ets1 and Notch at chromatin within enhancers to activate expression of known T-ALL oncogenes through its enhancer functions. Cdc73 also intersects with these factors within promoters to activate transcription of genes that are important for DNA repair and oxidative phosphorylation through its gene body functions. Consistently, Cdc73 deletion induced DNA damage and apoptosis and impaired mitochondrial function. The CDC73-induced DNA repair expression program co-opted by NOTCH1 is more highly expressed in T-ALL than in any other cancer. These data suggest that Cdc73 might induce a gene expression program that was eventually intersected and hijacked by oncogenic Notch to augment proliferation and mitigate the genotoxic and metabolic stresses of elevated Notch signaling. Our report supports studying factors such as CDC73 that intersect with Notch to derive a basic scientific understanding on how to combat Notch-dependent cancers without directly targeting the Notch complex.

BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment.

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

Inactivation of Capicua in adult mice causes T-cell lymphoblastic lymphoma.

CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4 Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancy.

Preclinical testing of CT1113, a novel USP28 inhibitor, for the treatment of T-cell acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a highly aggressive and heterogeneous lymphoid malignancy with poor prognosis in adult patients. Aberrant activation of the NOTCH1 signalling pathway is involved in the pathogenesis of over 60% of T-ALL cases. Ubiquitin-specific protease 28 (USP28) is a deubiquitinase known to regulate the stability of NOTCH1. Here, we report that genetic depletion of USP28 or using CT1113, a potent small molecule targeting USP28, can strongly destabilize NOTCH1 and inhibit the growth of T-ALL cells. Moreover, we show that USP28 also regulates the stability of sterol regulatory element binding protein 1 (SREBP1), which has been reported to mediate increased lipogenesis in tumour cells. As the most critical transcription factor involved in regulating lipogenesis, SREBP1 plays an important role in the metabolism of T-ALL. Therefore, USP28 may be a potential therapeutic target, and CT1113 may be a promising novel drug for T-ALL with or without mutant NOTCH1.

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

Epigenetically dysregulated NOTCH-Delta-HES signaling cascade can serve as a subtype classifier for acute lymphoblastic leukemia.

The NOTCH-Delta-HES signaling cascade is regarded as a double-edged sword owing to its dual tumor-suppressor and oncogenic roles, in different cellular environments. In the T-cells, it supports leukemogenesis by promoting differentiation while in B-cells, it controls leukemogenesis by inhibiting early differentiation/inducing growth arrest in the lead to apoptosis. The present study was undertaken to assess if this bi-faceted behavior of NOTCH family can be exploited as a diagnostic biomarker or subtype classifier of acute lymphoblastic leukemia (ALL). In this pursuit, expression of seven NOTCH cascade genes was analyzed in bone marrow (BM) biopsy and blood plasma (BP) of pediatric ALL patients using quantitative PCR (qPCR). Further, promoter DNA methylation status of the differentially expressed genes (DEGs) was assessed by methylation-specific qMSP and validated through bisulphite amplicon sequencing. Whereas hypermethylation of JAG1, DLL1, and HES-2, HES-4, and HES-5 was observed in all patients, NOTCH3 was found hypermethylated specifically in Pre-B ALL cases while DLL4 in Pre-T ALL cases. Aberrant DNA methylation strongly correlated with downregulated gene expression, which restored at complete remission stage as observed in "follow-up/post-treatment" subjects. The subtype-specific ROC curve analysis and Kaplan-Meier survival analysis predicted a clinically applicable diagnostic and prognostic potential of the panel. Moreover, the logistic regression model (Pre-B vs Pre-T ALL) was found to be the best-fitted model (McFadden's R2 = 0.28, F1 measure = 0.99). Whether analyzed in BM-aspirates or blood plasma, the NOTCH epigenetic signatures displayed comparable results (p < 0.001), advocating the potential of NOTCH-Delta-HES cascade, as a subtype classifier, in minimally invasive diagnosis of ALL.

LRH-1/NR5A2 interacts with the glucocorticoid receptor to regulate glucocorticoid resistance.

Nuclear receptors are transcription factors with important functions in a variety of physiological and pathological processes. Targeting glucocorticoid receptor (GR) activity using glucocorticoids is a cornerstone in the treatment of patients with T cell acute lymphoblastic leukemia (T-ALL), and resistance to GC-induced cell death is associated with poor outcome and a high risk for relapse. Next to ligand-binding, heterodimerization with other transcription factors presents an important mechanism for the regulation of GR activity. Here, we describe a GC-induced direct association of the Liver Receptor Homolog-1 (LRH-1) with the GR in the nucleus, which results in reciprocal inhibition of transcriptional activity. Pharmacological and molecular interference with LRH-1 impairs proliferation and survival in T-ALL and causes a profound sensitization to GC-induced cell death, even in GC-resistant T-ALL. Our data illustrate that direct interaction between GR and LRH-1 critically regulates glucocorticoid sensitivity in T-ALL opening up new perspectives for developing innovative therapeutic approaches to treat GC-resistant T-ALL.

Thymus-Brain Connections in T-Cell Acute Lymphoblastic Leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematologic disease caused by the transformation and uncontrolled proliferation of T-cell precursors. T-ALL is generally thought to originate in the thymus since lymphoblasts express phenotypic markers comparable to those described in thymocytes in distinct stages of development. Although around 50% of T-ALL patients present a thymic mass, T-ALL is characterized by peripheral blood and bone marrow involvement, and central nervous system (CNS) infiltration is one of the most severe complications of the disease. SUMMARY: The CNS invasion is related to the expression of specific adhesion molecules and receptors commonly expressed in developing T cells, such as L-selectin, CD44, integrins, and chemokine receptors. Furthermore, T-ALL blasts also express neurotransmitters, neuropeptides, and cognate receptors that are usually present in the CNS and can affect both the brain and thymus, participating in the crosstalk between the organs. KEY MESSAGES: This review discusses how the thymus-brain connections, mediated by innervation and common molecules and receptors, can impact the development and migration of T-ALL blasts, including CNS infiltration.

TET2 as a tumor suppressor and therapeutic target in T-cell acute lymphoblastic leukemia.

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy resulting from overproduction of immature T-cells in the thymus and is typified by widespread alterations in DNA methylation. As survival rates for relapsed T-ALL remain dismal (10 to 25%), development of targeted therapies to prevent relapse is key to improving prognosis. Whereas mutations in the DNA demethylating enzyme TET2 are frequent in adult T-cell malignancies, TET2 mutations in T-ALL are rare. Here, we analyzed RNA-sequencing data of 321 primary T-ALLs, 20 T-ALL cell lines, and 25 normal human tissues, revealing that TET2 is transcriptionally repressed or silenced in 71% and 17% of T-ALL, respectively. Furthermore, we show that TET2 silencing is often associated with hypermethylation of the TET2 promoter in primary T-ALL. Importantly, treatment with the DNA demethylating agent, 5-azacytidine (5-aza), was significantly more toxic to TET2-silenced T-ALL cells and resulted in stable re-expression of the TET2 gene. Additionally, 5-aza led to up-regulation of methylated genes and human endogenous retroviruses (HERVs), which was further enhanced by the addition of physiological levels of vitamin C, a potent enhancer of TET activity. Together, our results clearly identify 5-aza as a potential targeted therapy for TET2-silenced T-ALL.

Thymic-Epithelial-Cell-Dependent Microenvironment Influences Proliferation and Apoptosis of Leukemic Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological cancer characterized by the infiltration of immature T-cells in the bone marrow. Aberrant NOTCH signaling in T-ALL is mainly triggered by activating mutations of NOTCH1 and overexpression of NOTCH3, and rarely is it linked to NOTCH3-activating mutations. Besides the known critical role of NOTCH, the nature of intrathymic microenvironment-dependent mechanisms able to render immature thymocytes, presumably pre-leukemic cells, capable of escaping thymus retention and infiltrating the bone marrow is still unclear. An important challenge is understanding how leukemic cells shape their tumor microenvironment to increase their ability to infiltrate and survive within. Our previous data indicated that hyperactive NOTCH3 affects the CXCL12/CXCR4 system and may interfere with T-cell/stroma interactions within the thymus. This study aims to identify the biological effects of the reciprocal interactions between human leukemic cell lines and thymic epithelial cell (TEC)-derived soluble factors in modulating NOTCH signaling and survival programs of T-ALL cells and TECs. The overarching hypothesis is that this crosstalk can influence the progressive stages of T-cell development driving T-cell leukemia. Thus, we investigated the effect of extracellular space conditioned by T-ALL cell lines (Jurkat, TALL1, and Loucy) and TECs and studied their reciprocal regulation of cell cycle and survival. In support, we also detected metabolic changes as potential drivers of leukemic cell survival. Our studies could shed light on T-cell/stroma crosstalk to human leukemic cells and propose our culture system to test pharmacological treatment for T-ALL.

Targeted Metabolomics of Tissue and Plasma Identifies Biomarkers in Mice with NOTCH1-Dependent T-Cell Acute Lymphoblastic Leukemia.

While the genomics era has allowed remarkable advances in understanding the mechanisms driving the biology and pathogenesis of numerous blood cancers, including acute lymphoblastic leukemia (ALL), metabolic studies are still lagging, especially regarding how the metabolism differs between healthy and diseased individuals. T-cell ALL (T-ALL) is an aggressive hematological neoplasm deriving from the malignant transformation of T-cell progenitors characterized by frequent NOTCH1 pathway activation. The aim of our study was to characterize tumor and plasma metabolomes during T-ALL development using a NOTCH1-induced murine T-ALL model (ΔE-NOTCH1). In tissue, we found a significant metabolic shift with leukemia development, as metabolites linked to glycolysis (lactic acid) and Tricarboxylic acid cycle replenishment (succinic and malic acids) were elevated in NOTCH1 tumors, while metabolites associated with lipid oxidation (e.g., carnitine) as well as purine and pyrimidine metabolism were elevated in normal thymic tissue. Glycine, serine, and threonine metabolism, glutathione metabolism, as well as valine, leucine, and isoleucine biosynthesis were enriched pathways in tumor tissue. Phenylalanine and tyrosine metabolism was highly enriched in plasma from leukemia-bearing mice compared to healthy mice. Further, we identified a metabolic signature consisting of glycine, alanine, proline, 3-hydroxybutyrate, and glutamic acid as potential biomarkers for leukemia progression in plasma. Hopefully, the metabolic differences detected in our leukemia model will apply to humans and contribute to the development of metabolism-oriented therapeutic approaches.