Acute Lymphoblastic Leukemia Cells Research Articles

Therapeutic targeting of ACLY in T-ALL in vivo.

T-cell Acute Lymphoblastic Leukemia (T-ALL) is a hematological malignancy in need of novel therapeutic approaches. Here, we identify the ATP-citrate lyase ACLY as overexpressed and as a novel therapeutic target in T-ALL. To test the effects of ACLY in leukemia progression, we developed an isogenic model of NOTCH1-induced Acly conditional knockout leukemia. Importantly, we observed intrinsic antileukemic effects upon loss of ACLY, which further synergized with NOTCH1 inhibition in vivo. Metabolomic profiling upon ACLY loss revealed a metabolic crisis with reduced acetyl-CoA levels, as well as a decreased oxygen consumption rate. Gene expression profiling analyses showed that the transcriptional signature of ACLY loss very significantly correlates with the signature of MYC loss in vivo . Mechanistically, the decrease in acetyl-CoA led to reduced H3K27ac levels in Myc , resulting in transcriptional downregulation of Myc and drastically reduced MYC protein levels. Interestingly, our analyses also revealed a reciprocal relationship whereby ACLY itself is a direct transcriptional target of MYC, thus establishing a feedforward loop that is important for leukemia progression. Overall, our results identified a relevant ACLY-MYC axis and unveiled ACLY as a novel promising target for T-ALL treatment.

Abstract PO-001: GPR65-low tumor clones reprogram macrophage via VEGF signaling and confer antigen-independent CAR-T resistance

Abstract Chimeric antigen receptor (CAR)-T cell therapies can be curative for CD19+ hematological malignancies, though are limited by frequent patient relapse and response failures. Here, we identify GPR65 as a tumor-specific determinant of responsiveness to CAR-T cell therapy. In patients and an immune competent mouse model of B cell acute lymphoblastic leukemia (B-ALL) response to CAR-T cell therapy, high GPR65 is associated with a complete tumor response. In mice, GPR65 knockout (GPR65 KO) tumors are resistant to CAR-T cell therapy in vivo, without antigen loss. Single-cell network analyses reveal that GPR65 deficiency remodels tumor interactions with host macrophages, partially through increasing tumor VEGFA. This leads to increased macrophage numbers and preferential M2 macrophage polarization. Either depletion of host macrophages or deletion of VEGFA from GPR65 KO tumors restores responsiveness to CAR-T cell treatment. Anti-VEGFA therapy in combination with CAR-T cell administration also prolongs the survival of GPR65 KO tumor-bearing mice. Our results indicate that tumor GPR65 expression is important for B-ALL responses to CAR-T cell therapy through its impact on the tumor microenvironment and supports new approaches to identify and overcome CAR-T cell resistance. Citation Format: Jayadev Mavuluri. GPR65-low tumor clones reprogram macrophage via VEGF signaling and confer antigen-independent CAR-T resistance [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-001.

Characterization and Antileukemic Potential of Annona muricata Leaf Fractions on “MOLT-3” T-Cell Acute Lymphoblastic Leukemia Cells"

Results of this research demonstrate that leaf fractions of Annona muricata can cause the T-ALL cell line, molt-3, to undergo apoptosis. To find out what phytochemicals are in the Annona muricata leaf fraction (F4C), we used FTIR and GCMS in this investigation. Different functional groups were detected by FTIR findings, and different phytochemicals were detected by GCMS analysis in the F4C fraction. The cytotoxic efficacy of the F4C fraction against molt-3 cell lines were confirmed byMTT and SRB assays.The F4C treated cells terminated at the G0-G1 phase of the cell cycle, according to cell cycle analysis. It was determined that the substantial drop in MMP staining by JC-1 labelling corresponded to the loss of MMP. Anticancer efficacy against molt-3 cell line is thus demonstrated by Annona muricata leaf fractions.

Automation of 3D digital rolling circle amplification using a 3D-printed liquid handler

Automation of liquid handling is indispensable to improve throughput and reproducibility in biochemical assays. However, the incorporation of automated systems into laboratory workflows is often hindered by the high cost and complexity associated with building robotic liquid handlers. Here, we report a 3D-printed liquid handler based on a fluidic manifold, thereby obviating the need for complex robotic mechanisms. The fluidic manifold, termed a dispensing and aspirating (DA) device, comprises parallelized multi-pipette structures connected by distribution and aspiration channels, enabling the precise supply and removal of reagents, respectively. Leveraging the versatility of 3D printing, the DA device can be custom-designed and printed to fit specific applications. As a proof-of-principle, we engineered a 3D-printed liquid handler dedicated for 3D digital rolling circle amplification (4DRCA), an advanced biochemical assay involving multiple sample preparation steps such as antibody incubation, cell fixation, nucleic acid amplification, probe hybridization, and extensive washing. We demonstrate the efficacy of the 3D-printed liquid handler to automate the preparation of clinical samples for the simultaneous, in situ analysis of oncogenic protein and transcript markers in B-cell acute lymphoblastic leukemia cells using 4DRCA. This approach provides an effective and accessible solution for liquid handling automation, offering high throughput and reproducibility in biochemical assays.

Comparable efficacy of oral bendamustine versus intravenous administration in treating hematologic malignancies.

The purpose of this study was to analyze potential differences in antitumor efficacy and pharmacokinetics between intravenous (IV) bendamustine and a novel orally administered (PO) bendamustine agent that is utilizing the beneficial properties of superstaturated solid dispersions formulated in nanoparticles. Pharmacokinetics of IV versus PO bendamustine were determined by analysis of plasma samples collected from NSG mice treated with either IV or PO bendamustine. Plasma samples were analyzed using liquid chromatography-mass spectrometry following a liquid-liquid extraction to determine peak bendamustine concentration, area under the concentration-time curve, and the half-life in-vivo. In-vitro cytotoxicity of bendamustine against human non-Hodgkin Burkitt's Lymphoma (Raji), multiple myeloma (MM.1s), and B-cell acute lymphoblastic leukemia (RS4;11) cell lines was determined over time using MTS assays. Luciferase-tagged versions of the aforementioned cell lines were used to determine in-vivo bendamustine cytotoxicity of IV versus PO bendamustine at two different doses. Bendamustine at a high dose in-vitro causes cell death. There was no significant difference in antitumor activity between IV and novel PO bendamustine at a physiologically relevant concentration in all three xenograft models. In-vivo pharmacokinetics showed the oral bioavailability of bendamustine in mice to be 51.4%. The novel oral bendamustine agent tested exhibits good oral bioavailability and systemic exposure for in-vivo antitumor efficacy comparable to IV bendamustine. An oral bendamustine formulation offers exciting clinical potential as an additional method of administration for bendamustine and warrants further evaluation in clinical studies.

Conserved epigenetic hallmarks of T cell aging during immunity and malignancy

Chronological aging correlates with epigenetic modifications at specific loci, calibrated to species lifespan. Such ‘epigenetic clocks’ appear conserved among mammals, but whether they are cell autonomous and restricted by maximal organismal lifespan remains unknown. We used a multilifetime murine model of repeat vaccination and memory T cell transplantation to test whether epigenetic aging tracks with cellular replication and if such clocks continue ‘counting’ beyond species lifespan. Here we found that memory T cell epigenetic clocks tick independently of host age and continue through four lifetimes. Instead of recording chronological time, T cells recorded proliferative experience through modification of cell cycle regulatory genes. Applying this epigenetic profile across a range of human T cell contexts, we found that naive T cells appeared ‘young’ regardless of organism age, while in pediatric patients, T cell acute lymphoblastic leukemia appeared to have epigenetically aged for up to 200 years. Thus, T cell epigenetic clocks measure replicative history and can continue to accumulate well-beyond organismal lifespan.

Mutation and cell state compatibility is required and targetable in Ph+ acute lymphoblastic leukemia minimal residual disease.

Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the availability of tyrosine kinase inhibitors (TKIs) with broad activity against resistance mutants. The mechanisms that drive persistence within minimal residual disease (MRD) remain poorly understood and therefore untargeted. Utilizing 13 patient-derived xenograft (PDX) models and clinical trial specimens of Ph+ ALL, we examined how genetic and transcriptional features co-evolve to drive progression during prolonged TKI response. Our work reveals a landscape of cooperative mutational and transcriptional escape mechanisms that differ from those causing resistance to first generation TKIs. By analyzing MRD during remission, we show that the same resistance mutation can either increase or decrease cellular fitness depending on transcriptional state. We further demonstrate that directly targeting transcriptional state-associated vulnerabilities at MRD can overcome BCR::ABL1 independence, suggesting a new paradigm for rationally eradicating MRD prior to relapse. Finally, we illustrate how cell mass measurements of leukemia cells can be used to rapidly monitor dominant transcriptional features of Ph+ ALL to help rationally guide therapeutic selection from low-input samples.

Specificity protein 1/3 regulate T-cell acute lymphoblastic leukemia cell proliferation and apoptosis through β-catenin by acting as targets of miR-495-3p.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic heterogeneous disease. This study explored the mechanism of specificity protein 1/3 (Sp1/3) in T-ALL cells through β-catenin by acting as targets of miR-495-3p. Expression levels of miR-495-3p, Sp1, Sp3, and β-catenin in the serum from T-ALL children patients, healthy controls, and the T-ALL cell lines were measured. The cell proliferation ability and apoptosis rate were detected. Levels of proliferation-related proteins proliferating cell nuclear antigen (PCNA)/cyclinD1 and apoptosis-related proteins B-cell lymphoma-2 associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) were determined. The binding of Sp1/3 and β-catenin promoter and the targeted relationship between miR-495-3p with Sp1/3 were analyzed. Sp1/3 were upregulated in CD4+ T-cells in T-ALL and were linked with leukocyte count and risk classification. Sp1/3 interference prevented proliferation and promoted apoptosis in T-ALL cells. Sp1/3 transcription factors activated β-catenin expression. Sp1/3 enhanced T-ALL cell proliferation by facilitating β-catenin expression. miR-495-3p targeted and repressed Sp1/3 expressions. miR-495-3p overexpression inhibited T-ALL cell proliferation and promoted apoptosis. Conjointly, Sp1/3, as targets of miR-495-3p limit apoptosis and promote proliferation in T-ALL cells by promoting β-catenin expression.

Glucocorticoids paradoxically promote steroid resistance in B cell acute lymphoblastic leukemia through CXCR4/PLC signaling

Glucocorticoid (GC) resistance in childhood relapsed B-cell acute lymphoblastic leukemia (B-ALL) represents an important challenge. Despite decades of clinical use, the mechanisms underlying resistance remain poorly understood. Here, we report that in B-ALL, GC paradoxically induce their own resistance by activating a phospholipase C (PLC)-mediated cell survival pathway through the chemokine receptor, CXCR4. We identify PLC as aberrantly activated in GC-resistant B-ALL and its inhibition is able to induce cell death by compromising several transcriptional programs. Mechanistically, dexamethasone (Dex) provokes CXCR4 signaling, resulting in the activation of PLC-dependent Ca2+ and protein kinase C signaling pathways, which curtail anticancer activity. Treatment with a CXCR4 antagonist or a PLC inhibitor improves survival of Dex-treated NSG mice in vivo. CXCR4/PLC axis inhibition significantly reverses Dex resistance in B-ALL cell lines (in vitro and in vivo) and cells from Dex resistant ALL patients. Our study identifies how activation of the PLC signalosome in B-ALL by Dex limits the upfront efficacy of this chemotherapeutic agent.

Perinatal thymic-derived CD8αβ-expressing γδ T cells are innate IFN-γ producers that expand in IL-7R–STAT5B-driven neoplasms

The contribution of γδ T cells to immune responses is associated with rapid secretion of interferon-γ (IFN-γ). Here, we show a perinatal thymic wave of innate IFN-γ-producing γδ T cells that express CD8αβ heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αβ+ γδ T cell development is directed by low T cell receptor signaling and through provision of interleukin (IL)-4 and IL-7. This population is pathologically relevant as overactive, or constitutive, IL-7R–STAT5B signaling promotes a supraphysiological accumulation of CD8αβ+ γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αβ+ γδ T cells define a distinct subset of human T cell acute lymphoblastic leukemia pediatric patients. This work characterizes the normal and malignant development of CD8αβ+ γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer.

Overexpression of PTPN21 promotes proliferation of EGF-stimulated acute lymphoblastic leukemia cells via the MAPK signaling pathways

ABSTRACT Objectives This study aims to investigate the role of excessive Protein Tyrosine Phosphatase Non-Receptor Type 21 (PTPN21) in the proliferation of Acute Lymphoblastic Leukemia (ALL) cells with EGF stimulation. Methods PTPN21 was overexpressed in ALL cell lines by lentiviral transfection. Apoptosis was assayed by Annexin V/7-AAD staining. The proliferation and cell cycle of EGF-treated ALL cells were assessed by MTT and Ki-67/7-AAD staining respectively. The phosphorylation of Src tyrosine kinase and mediators of distinct MAPK pathways were assessed by Western blot. Results Overexpression of PTPN21 had minimal effect on the apoptosis of ALL cells, but significantly promoted the proliferation and cell cycle progression of ALL cells stimulated with EGF. The activity of Src tyrosine kinase and the MAPK pathways was elevated. Inhibition of MAPK pathways by specific inhibitors mitigated this pro-proliferative effect of excessive PTPN21 on EGF-stimulated ALL cells. Conclusion PTPN21 may facilitate ALL progression by promoting cell proliferation via the Src/MAPK signaling pathways.

Dihydroartemisinin induces ferroptosis in T cell acute lymphoblastic leukemia cells by downregulating SLC7A11 and activating the ATF4‑CHOP signaling pathway

Dihydroartemisinin induces ferroptosis in T cell acute lymphoblastic leukemia cells by downregulating SLC7A11 and activating the ATF4‑CHOP signaling pathway

A Capillary-Force-Driven, Single-Cell Transfer Method for Studying Rare Cells.

The characterization of individual cells within heterogeneous populations (e.g., rare tumor cells in healthy blood cells) has a great impact on biomedical research. To investigate the properties of these specific cells, such as genetic biomarkers and/or phenotypic characteristics, methods are often developed for isolating rare cells among a large number of background cells before studying their genetic makeup and others. Prior to using real-world samples, these methods are often evaluated and validated by spiking cells of interest (e.g., tumor cells) into a sample matrix (e.g., healthy blood) as model samples. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. People often circumvent the problem by diluting a solution of high-concentration cells, but the concentration becomes inaccurate after series dilution due to the fact that a cell suspension solution can be inhomogeneous, especially when the cell concentration is very low. We report on an alternative method for low-cost, accurate, and reproducible low-concentration cell spiking without the use of external pumping systems. By inducing a capillary force from sudden pressure drops, a small portion of the cellular membrane was aspirated into the reservoir tip, allowing for non-destructive single-cell transfer. We investigated the surface membrane tensions induced by cellular aspiration and studied a range of tip/tumor cell diameter combinations, ensuring that our method does not affect cell viability. In addition, we performed single-cell capture and transfer control experiments using human acute lymphoblastic leukemia cells (CCRF-CEM) to develop calibrated data for the general production of low-concentration samples. Finally, we performed affinity-based tumor cell isolation using this method to generate accurate concentrations ranging from 1 to 15 cells/mL.

A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.

We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

Disruption of the Hippo pathway promotes the proliferation of childhood acute lymphoblastic leukemia cells, inhibits apoptosis and chemosensitivity

ABSTRACT Background Despite advancements in chemotherapy and stem cell transplantation, the recurrence and chemoresistance of childhood acute lymphoblastic leukemia (cALL) remain a significant challenge, thus indicating the need for novel therapeutic targets. Research design and methods The protein levels of YAP1, p-YAP1, TAZ, and Cyr61 of cALL patients and healthy volunteers were measured by western blot analysis. Then the leukemic cell line SUP-B15 was transfected with sh-YAP1 and pcDNA3.1-YAP1 to knockdown or overexpress YAP1. The viability, chemosensitivity, apoptosis, migration, and invasion of SUP-B15 cells were determined by MTT, flow cytometry, and Transwell assay. Results The cALL patients had higher YAP1, TAZ, and Cyr61 protein expression and lower p-YAP1 protein expression in bone marrow tissues compared with healthy volunteers (p < 0.01). In SUP-B15 cells, YAP1 knockdown upregulated p-YAP1 protein expression (p < 0.01) and downregulated TAZ and Cyr61 protein expression (p < 0.01). In addition, knocking down YAP1 significantly inhibited cell viability, migration, and invasion, and induced apoptosis (p < 0.01). YAP1 knockdown also reduced the IC50 value following treatment with vincristine, daunorubicin, cyclophosphamide, and dexamethasone (p < 0.05). Conclusions Disruption of the Hippo pathway attenuates the development of cALL by promoting cell proliferation while suppressing apoptosis and drug sensitivity.

Expression of transcriptional repressors of ZBTB genes in acute lymphoblastic leukemia cell lines treated with prednisolone

Expression of transcriptional repressors of ZBTB genes in acute lymphoblastic leukemia cell lines treated with prednisolone

Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing leukemia cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion.

Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approximately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. CD20 express on 30 to 50% of B-ALL, which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy. Adding Rituximab to chemotherapy protocol had been demonstrated to improve the outcome for CD20-positive ALL. However, rare study explored the influence of Rituximab combined with CAR-T therapy. We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T therapy, all of whom had failed multiple lines of therapy. Before CAR-T infusion, we administered Rituximab to 10 patients with high CD20 expression at a dose of 375mg/m2 for 1 day. Meanwhile, we selected 10 patients with the comparable features who underwent CAR-T treatment without Rituximab in the same period as the control group. In vitro, the surface molecule expression and killing of CAR-T post Rituximab-treated B-ALL cells co-incubated with CAR-T cells were detected by flow cytometry. The median follow-up of Rituximab and Control groups were 29.27 and 9.83 months. We found that adding Rituximab may confer a favorable prognosis compared with Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates both were longer in the Rituximab group (90% vs. 26.7%, p=0.0342; 41.7% vs. 25%, p=0.308). In vitro, we observed that Rituximab-treated tumour cells are more sensitive to CAR-T killing and a broad range of cytokines and chemokines were produced when Rituximab-treated Nalm-6 cells co-cultured with 19-22CAR-T cells, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). To investigate whether Rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells repeatedly in vitro with Rituximab-treated Nalm-6 to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of exhaustion molecules (LAG-3, PD-1, TIM-3) on CAR-T cells were significantly lower in the Rituximab group than in the Control group. Rituximab combined with CAR-T therapy is effective for improving the long-term prognosis of B-ALL patients who have failed multiple lines of therapy. In vitro, we observed that rituximab potentially improves CAR-T efficacy by sensitizing ALL to CART-mediated cytotoxicity and reducing CAR-T exhaustion.

Plasma microbial cell-free DNA following chimeric antigen receptor T cell therapy in pediatric patients with relapsed/refractory leukemia.

Chimeric antigen receptor (CAR) T cell therapy is a promising treatment for pediatric patients with relapsed or refractory B cell acute lymphoblastic leukemia (R/R B ALL). Cytokine release syndrome (CRS) is a common toxicity after CAR T cell therapy and fever is often the first symptom. Differentiating CRS from infection after CAR T cell therapy can be challenging. Plasma microbial cell free DNA (mcfDNA) is a novel diagnostic tool which allows for qualitative and quantitative assessment of over 1000 organisms. This pilot study sought to characterize mcfDNA results in pediatric patients with R/R B ALL in the first 2 months after CAR T cell therapy.

Chimeric antigen receptor (CAR) modified T Cells in acute myeloid leukemia: limitations and expectations.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis despite the advent of novel therapies. Consequently, a major need exists for new therapeutic options, particularly for patients with relapsed/refractory (R/R) AML. In recent years, it has been possible to individualize the treatment of a subgroup of patients, particularly with the emergence of multiple targeted therapies. Nonetheless, a considerable number of patients remain without therapeutic options, and overall prognosis remains poor because of a high rate of disease relapse. In this sense, cellular therapies, especially chimeric antigen receptor (CAR)-T cell therapy, have dramatically shifted the therapeutic options for other hematologic malignancies, such as diffuse large B cell lymphoma and acute lymphoblastic leukemia. In contrast, effectively treating AML with CAR-based immunotherapy poses major biological and clinical challenges, most of them derived from the unmet need to identify target antigens with expression restricted to the AML blast without compromising the viability of the normal hematopoietic stem cell counterpart. Although those limitations have hampered CAR-T cell therapy translation to the clinic, there are several clinical trials where target antigens, such as CD123, CLL-1 or CD33 are being used to treat AML patients showing promising results. Moreover, there are continuing efforts to enhance the specificity and efficacy of CAR-T cell therapy in AML. These endeavors encompass the exploration of novel avenues, including the development of dual CAR-T cells and next-generation CAR-T cells, as well as the utilization of gene editing tools to mitigate off-tumor toxicities. In this review, we will summarize the ongoing clinical studies and the early clinical results reported with CAR-T cells in AML, as well as highlight CAR-T cell limitations and the most recent approaches to overcome these barriers. We will also discuss how and when CAR-T cells should be used in the context of AML.

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.