T-cell Progenitors Research Articles

The JAK3Q988P mutation reveals oncogenic potential and resistance to ruxolitinib.

T-cell acute lymphoblastic leukemia (T-ALL) arises from the malignant transformation of T-cell progenitors at various differentiation stages. Given that patients who relapse have a dismal prognosis, there is an urgent need to identify the molecular alterations that are present in such patients and promote leukemogenesis to implement personalized therapies with higher efficacy and fewer adverse effects. In the present manuscript, we identified the JAK3Q988P mutation in a T-ALL patient who did not achieve a durable response after the conventional treatment and whose tumor cells at relapse presented constitutive activation of the JAK/STAT pathway. Although JAK3Q988P has been previously identified in T-ALL patients from different studies, the functional consequences exerted by this mutation remain unexplored. Through the combination of different hematopoietic cellular models, we functionally characterize JAK3Q988P as an oncogenic mutation that contributes to leukemogenesis. Notably, JAK3Q988P not only promotes constitutive activation of the JAK/STAT pathway in the absence of cytokines and growth factors, as is the case for other JAK3 mutations that have been functionally characterized as oncogenic, but also functions independently of JAK1 and IL2RG, resulting in high oncogenic potential as well as resistance to ruxolitinib. Our results indicate that ruxolitinib may not be efficient for future patients bearing the JAK3Q988P mutation who instead may obtain greater benefits from treatments involving other pharmacological inhibitors such as tofacitinib.

BH3 profiling identifies BCL-2 dependence in adult patients with early T-cell progenitor acute lymphoblastic leukemia.

BH3 profiling identifies BCL-2 dependence in adult patients with early T-cell progenitor acute lymphoblastic leukemia.

A Comprehensive Analysis of the Expression Profiles of KCTD Proteins in Acute Lymphoblastic Leukemia: Evidence of Selective Expression of KCTD1 in T-ALL.

Acute leukemia is the most common pediatric cancer. In most cases, this disease results from the malignant transformation of either the B-cell (B-ALL) or, less frequently, T-cell progenitors (T-ALL). Recently, a marked overexpression of KCTD15, a member of the emerging class of the potassium (K) channel tetramerization domain-containing proteins (KCTDs) has been detected in both patients and continuous cell lines as in vitro model systems. Because there is growing evidence of the key, yet diversified, roles played by KCTDs in cancers, we here report an exhaustive analysis of their expression profiles in both B-ALL and T-ALL patients. Although for most KCTDs, no significant alterations were found in these pathological states, for some members of the family, significant up- and down-regulations were detected in comparison with the values found in healthy subjects in the transcriptome analysis. Among these, particularly relevant is the upregulation of the closely related KCTD1 and KCTD15 in T-ALL patients. Interestingly, KCTD1 is barely expressed in both unaffected controls and B-ALL patients. Therefore, not only does this analysis represent the first study in which the dysregulation of all KCTDs is simultaneously evaluated in specific pathological contexts, but it also provides a promising T-ALL biomarker that could be suitable for clinical applications.

The initial age-associated decline in early T-cell progenitors reflects fewer pre-thymic progenitors and altered signals in the bone marrow and thymus microenvironments.

Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.

Abstract 6396: BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia

Abstract Background: The chronic lymphocytic leukemia (CLL) tumor microenvironment (TME) is laden with hyporesponsive T-cells that permit disease persistence. Yet, redundant TME immunosuppressive mechanisms and epigenetic maintenance of T-cell exhaustion limit the efficacy of T-cell targeted therapies in CLL. Bromodomain and extra-terminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T-cell function and differentiation. We hypothesize that blocking BET protein function can reverse T-cell exhaustion to yield durable tumor elimination in CLL. Methods: WT C57BL/6 mice were engrafted with Eμ-TCL1 spleen-derived lymphocytes, then treated daily with the novel pan-BET inhibitor, OPN-51107 (OPN5; 20mg/kg PO) for up to 4 weeks. Splenic gene expression was evaluated with the NanoString PanCancer iO360 panel. T-cell differentiation status, immune inhibitory receptor (IR) expression, proliferation (72 h ex vivo α-CD3/α-CD28 stimulation), and cytokine production (6 h ex vivo PMA/ionomycin stimulation) was measured via flow cytometry. CLL patient and healthy donor PBMCs were used for validation studies and to assess T-cell transcription factor (TF) expression via flow cytometry. Evaluation of BRD4 occupancy at select T-cell TFs via ChIP qPCR is ongoing. Results: OPN5 significantly increased cytotoxic cell signatures and reduced exhaustion-associated cell signatures in leukemic mice through inhibition of T-cell exhaustion signaling, as well as activation of Th1, natural killer cell, and IL-7 signaling pathways. Correspondingly, T-cells from OPN5-treated mice demonstrated greater ex vivo proliferative capacity and effector response to stimuli. A greater proportion of CD8+ T-cells from OPN5-treated mice were classified as naïve, and OPN5 significantly reduced KLRG1 expression on antigen-experienced CD8+ T-cells. Importantly, OPN5 curtailed IR co-expression (PD-1, PD-L1, VISTA, CD244, CD160, and LAG3) on splenic T-cells. These findings were confirmed with primary CLL cells ex vivo. OPN5 also impaired expression of terminal differentiation-associated TFs in CLL patient-derived T-cells, enriching for a TCF1+ progenitor T-cell population. While BTK inhibitors are known to similarly improve T-cell function in CLL, ibrutinib treatment was inadequate to revert CLL T-cell terminal differentiation. Future ATAC-sequencing analysis will inform how BET inhibition alleviates exhaustion-associated chromatin organization in CLL T-cells. Conclusion: BET inhibition dismantles immunosuppressive mechanisms in the CLL TME, alleviating CLL-induced T-cell dysfunction and terminal differentiation. These findings suggest that BET inhibition may be a useful component of combination strategies for the treatment of CLL to yield lasting anti-cancer immune memory and prevent relapsed/refractory disease. Citation Format: Audrey L. Smith, Alexandria P. Eiken, Sydney A. Skupa, Christopher R. D'Angelo, Avyakta Kallam, Matthew A. Lunning, Gregory Bociek, Julie M. Vose, Ben Powell, Gideon Bollag, Dalia El-Gamal. BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6396.

Multi-omics integration reveals potential stage-specific druggable targets in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL), a heterogeneous hematological malignancy, is caused by the developmental arrest of normal T-cell progenitors. The development of targeted therapeutic regimens is impeded by poor knowledge of the stage-specific aberrances in this disease. In this study, we performed multi-omics integration analysis, which included mRNA expression, chromatin accessibility, and gene-dependency database analyses, to identify potential stage-specific druggable targets and repositioned drugs for this disease. This multi-omics integration helped identify 29 potential pathological genes for T-ALL. These genes exhibited tissue-specific expression profiles and were enriched in the cell cycle, hematopoietic stem cell differentiation, and the AMPK signaling pathway. Of these, four known druggable targets (CDK6, TUBA1A, TUBB, and TYMS) showed dysregulated and stage-specific expression in malignant T cells and may serve as stage-specific targets in T-ALL. The TUBA1A expression level was higher in the early T cell precursor (ETP)-ALL cells, while TUBB and TYMS were mainly highly expressed in malignant T cells arrested at the CD4 and CD8 double-positive or single-positive stage. CDK6 exhibited a U-shaped expression pattern in malignant T cells along the naïve to maturation stages. Furthermore, mebendazole and gemcitabine, which target TUBA1A and TYMS, respectively, exerted stage-specific inhibitory effects on T-ALL cell lines, indicating their potential stage-specific antileukemic role in T-ALL. Collectively, our findings might aid in identifying potential stage-specific druggable targets and are promising for achieving more precise therapeutic strategies for T-ALL.

RNA helicase DHX15 exemplifies a unique dependency in acute leukemia.

RNA-binding proteins (RBP) have emerged as essential regulators that control gene expression and modulate multiple cancer traits. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy derived from transformation of T-cell progenitors that normally undergo discrete steps of differentiation in the thymus. The implications of essential RBP during T-cell neoplastic transformation remain largely unclear. Systematic evaluation of RBP identifies RNA helicase DHX15, which facilitates the disassembly of the spliceosome and release of lariat introns, as a T-ALL dependency factor. Functional analysis using multiple murine T-ALL models demonstrates the essential importance of DHX15 in tumor cell survival and leukemogenesis. Moreover, single-cell transcriptomics reveals that DHX15 depletion in T-cell progenitors hinders burst proliferation during the transition from doublenegative to double-positive cells (CD4-CD8- to CD4+CD8+). Mechanistically, abrogation of DHX15 perturbs RNA splicing and leads to diminished levels of SLC7A6 and SLC38A5 transcripts due to intron retention, thereby suppressing glutamine import and mTORC1 activity. We further propose a DHX15 signature modulator drug ciclopirox and demonstrate that it has prominent anti-T-ALL efficacy. Collectively, our data highlight the functional contribution of DHX15 to leukemogenesis through regulation of established oncogenic pathways. These findings also suggest a promising therapeutic approach, i.e., splicing perturbation by targeting spliceosome disassembly, may achieve considerable anti-tumor efficacy.

KDM6B protects T-ALL cells from NOTCH1-induced oncogenic stress.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors. While activating NOTCH1 mutations are the dominant genetic drivers of T-ALL, epigenetic dysfunction plays a central role in the pathology of T-ALL and can provide alternative mechanisms to oncogenesis in lieu of or in combination with genetic mutations. The histone demethylase enzyme KDM6A (UTX) is also recurrently mutated in T-ALL patients and functions as a tumor suppressor. However, its gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed, suggesting it may be necessary for sustaining the disease. Here, we used mouse and human T-ALL models to show that KDM6B is required for T-ALL development and maintenance. Using NOTCH1 gain-of-function retroviral models, mouse cells genetically deficient for Kdm6b were unable to propagate T-ALL. Inactivating KDM6B in human T-ALL patient cells by CRISPR/Cas9 showed KDM6B-targeted cells were significantly outcompeted over time. The dependence of T-ALL cells on KDM6B was proportional to the oncogenic strength of NOTCH1 mutation, with KDM6B required to prevent stress-induced apoptosis from strong NOTCH1 signaling. These studies identify a crucial role for KDM6B in sustaining NOTCH1-driven T-ALL and implicate KDM6B as a novel therapeutic target in these patients.

Intrathymic AAV delivery results in therapeutic site-specific integration at TCR loci in mice

AbstractAdeno-associated virus (AAV) vectors have been successfully exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector, but it has been shown to integrate within the host cell genome after the generation of double-strand DNA breaks or nicks. Although AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T-cell progenitors and long-term T-cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T cells and compared these with liver and brain tissue from treated mice. Notably, we found that although AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T cells were clustered within the T-cell receptor α, β, and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAGs) during variable, diversity, and joining recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells.

Graft-versus-host disease is locally maintained in target tissues by resident progenitor-like T cells

In allogeneic hematopoietic stem cell transplantation, donor αβ Tcells attack recipient tissues, causing graft-versus-host disease (GVHD), a major cause of morbidity and mortality. A central question has been how GVHD is sustained despite Tcell exhaustion from chronic antigen stimulation. The current model for GVHD holds that disease is maintained through the continued recruitment of alloreactive effectors from blood into affected tissues. Here, we show, using multiple approaches including parabiosis of mice with GVHD, that GVHD is instead primarily maintained locally within diseased tissues. By tracking 1,203 alloreactive Tcell clones, we fitted a mathematical model predicting that within each tissue a small number of progenitor Tcells maintain a larger effector pool. Consistent with this, we identified a tissue-resident TCF-1+ subpopulation that preferentially engrafted, expanded, and differentiated into effectors upon adoptive transfer. These results suggest that therapies targeting affected tissues and progenitor Tcells within them would be effective.

Generation of CD34+CD43+ Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells

Immunotherapy using primary T cells has revolutionized medical care in some pathologies in recent years, but limitations associated to challenging cell genome edition, insufficient cell number production, the use of only autologous cells, and the lack of product standardization have limited its clinical use. The alternative use of T cells generated in vitro from human pluripotent stem cells (hPSCs) offers great advantages by providing a self-renewing source of T cells that can be readily genetically modified and facilitate the use of standardized universal off-the-shelf allogeneic cell products and rapid clinical access. However, despite their potential, a better understanding of the feasibility and functionality of T cells differentiated from hPSCs is necessary before moving into clinical settings. In this study, we generated human-induced pluripotent stem cells from T cells (T-iPSCs), allowing for the preservation of already recombined TCR, with the same properties as human embryonic stem cells (hESCs). Based on these cells, we differentiated, with high efficiency, hematopoietic progenitor stem cells (HPSCs) capable of self-renewal and differentiation into any cell blood type, in addition to DN3a thymic progenitors from several T-iPSC lines. In order to better comprehend the differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had very similar profiles to cord blood hematopoietic stem cells (HSCs). Furthermore, differentiated T-cell progenitors had a similar profile to thymocytes at the DN3a stage of thymic lymphopoiesis. Therefore, utilizing this approach, we were able to regenerate precursors of therapeutic human T cells in order to potentially treat a wide range of diseases.

High-Endothelial Venules Promote Permissive T-cell Niches after Immunotherapy.

Tumoral high-endothelial venule (TU-HEV) formation after antiangiogenic immunotherapy generates niches for progenitor T-cell expansion and differentiation into effector T cells.

Mutational analysis of ribosomal proteins in a cohort of pediatric patients with T-cell acute lymphoblastic leukemia reveals Q123R, a novel mutation in RPL10.

T-cell acute lymphoblastic leukemia (T-ALL) is a subtype of ALL involving the malignant expansion of T-cell progenitors. It is driven by a number of different possible genetic lesions, including mutations in genes encoding for ribosomal proteins (RPs). These are structural constituents of ribosomes, ubiquitous effectors of protein synthesis. Albeit the R98S mutation in RPL10, recurring with a higher frequency among RP mutations, has been extensively studied, less is known about the contribution of mutations occurring in other RPs. Alterations affecting translational machinery may not be well tolerated by cells, and there may be a selective pressure that determines the emergence of mutations with a compensatory effect. To explore this hypothesis, we sequenced the exomes of a cohort of 37 pediatric patients affected by T-ALL, and analyzed them to explore the co-occurrence of mutations in genes involved in ribosome biogenesis (including RPs) and translational control, and in known T-ALL driver genes. We found that some of the mutations in these sub-classes of genes tend to cluster together in different patients, indicating that their co-occurrence may confer some kind of advantage to leukemia cells. In addition, our sequencing highlighted the presence of a novel mutation in RPL10, namely the Q123R, which we found associated with a defect in protein synthesis. Our findings indicate that genetic alterations involving ribosome biogenesis and translational control should be carefully considered in the context of precision medicine in T-ALL.

DNMT3A Limits Myeloid Signaling Responses in Committed T Cells during Normal and Leukemic Development

T-cell development and lineage commitment are temporally protracted processes in which the interplay between transcription factors and epigenetic modulators coordinate the sequential exclusion of alternative fates and acquisition of specialized T-cell functions. Alterations during this process can lead to diseases such as T-cell acute lymphoblastic leukemia (T-ALL). While many efforts have focused on the transcription factors which drive normal development and leukemogenesis, the underlying epigenetic constraints remain poorly understood. Further, characterization of the role of particular factors during early stages of human T-cell development is limited by difficulty in obtaining and manipulating primary thymic tissue. Here, we characterize T-cell development as modeled in a chemically defined in vitro differentiation system using primary human CD34+ cord blood (CB) as input material (StemSpan T Cell Generation Kit, Stem Cell Technologies). This culture system yields large numbers of genetically manipulable cells that recapitulate aspects of normal intrathymic development based on immunophenotype, gene expression profiling, T-cell receptor rearrangement, and lineage restriction. DNMT3A is recurrently mutated in human T-ALL with identified variants thought to result in protein loss-of-function (LOF). As well, DNMT3A mutations are enriched in ETP-TALL, a phenotypic subtype with transcriptional signature resembling stem/myeloid-like progenitors. We thus sought to explore the role of DNMT3A in early stages of T cell lineage commitment using the in vitro CB differentiation system in combination with lentiviral shRNA-mediated knockdown (KD) and CRISPR/Cas9-mediated knockout (KO) to mimic DNMT3A LOF mutations. To assess durability of commitment to the T lineage, we transferred cells at the post-commitment CD44- stage from culture conditions supporting T cell differentiation to conditions favoring myeloid differentiation (StemSpan Myeloid Expansion Supplements I and II for granulocytes and monocytes, respectively). We found using clonogenic cell growth assays performed at limiting dilution that T cells with DNMT3A KD/KO were able to grow in myeloid media and expressed an aberrant T/myeloid phenotype at significantly higher frequency as compared to negative controls. Interestingly, this lineage switching activity was nearly exclusively represented in a small subset of cells that expressed myeloid growth factor receptors on the cell surface which were present in both KD and control in vitro derived T cells, as well as in normal thymus at similar frequencies. Minor subsets of myeloid receptor positive cells are also identifiable in established human T-ALL cell lines, and similar to in vitro derived T cells, the receptor positive subset of DNMT3A KD cells showed preferential growth in response to stimulation with their cognate ligands. Phosphoflow assays demonstrated that KD and non-silencing/negative control cells showed similar phosphoprotein signaling responses upon myeloid growth factor stimulation, suggesting that growth responses in KD cells are enacted at the DNA level, presumably by reduced DNA methylation at target gene promoters. These results support a working model in which DNMT3A loss in T-cell progenitors restores their ability to respond positively to myeloid growth factor stimulation both in terms of cell growth and myeloid marker expression. Of note, interrogation of clonal progeny from individual DNMT3A KO cells reveal no evidence of differentiation delay or arrest under T-tropic culture conditions, suggesting that responses to myeloid-tropic conditions are due to reprogramming of T-committed cells rather than increased persistence of uncommitted cells. These processes also appear to be operative in established T-ALL cells, suggesting that DNMT3A LOF mutation may afford T-ALL cells the ability to exploit alternate myeloid factor replete niches in the body.

DNMT3A Mutations Enhance T-ALL Survival, Which Is Reversed By JAK/STAT Inhibition

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm of T-cell progenitors, which accounts for approximately 15% and 25% of pediatric and adult acute lymphoblastic leukemia (ALL) cases respectively. In T-ALL, accumulation of genomic abnormalities often leads to the aberrant expression of select groups of transcription factors that result in increased proliferation, cell survival and impaired differentiation of T-cell progenitors, which give rise to this disease The Notch signaling pathway is essential for T-lymphopoiesis and T-cell differentiation from thymic precursors. Moreover, gain-of-function mutations in this pathway act as the major genetic lesions that drive T-ALL development (over 60% of adult cases). The de novo DNA methyltransferase enzyme (DNMT3A) is one of the three genes in mammals encoding for enzymes with DNA methyltransferase activity and mutations in this gene are frequently associated with NOTCH1 mutations in adult T-ALL. Interestingly, DNMT3A mutations are rare in pediatric and adolescent T-ALL. Prognosis of pediatric and adolescent T-ALL cases has seen major improvements during recent years with the introduction of high-dose, multi-agent chemotherapy regimens which has not carried over to adult T-ALL patients. These factors suggest the involvement of genetic differences between adult and pediatric T-ALL patients for the differential clinical outcomes Previous work in our lab has shown that mouse T-ALL cells genetically deficient for Dnmt3a are less apoptotic and have increased JAK/STAT signaling. Here, we demonstrate that DNMT3A-mutant human T-ALL patient derived xenografts (PDXs) are resistant to the chemotherapeutic drugs dexamethasone (DEX) and doxorubicin compared to T-ALL patient specimens with wild-type DNMT3A. However, when primary DNMT3A-mutant human T-ALL cells were treated with dexamethasone or doxorubicin in combination with the JAK/STAT inhibitor ruxolitinib (RUX), we observed the survival benefit of DNMT3A-mutant T-ALL cells were mitigated, suggesting enhanced JAK/STAT signaling promotes survival in T-ALL patient cells with DNMT3A mutations. To investigate the genes and pathways that endows DNMT3A-mutant T-ALL cells chemoresistance, gene expression profiling was performed by RNA-seq on DNMT3A-mutant, and wild-type human T-ALL cells treated with DEX+RUX. Gene set enrichment analysis (GSEA) showed that MTORC1, IL2-STAT5, E2F and G2M pathways were significantly downregulated in DEX+RUX treated DNMT3A-mutant PDX samples. E2F and G2M pathways are associated with cell cycle progression and further analysis of these two pathways revealed BIRC5 as a common gene that is downregulated in both these pathways following DEX+RUX treatment. Furthermore, our RNA-seq analysis demonstrated that the expression of BIRC5 in DNMT3A-mutant PDXs is downregulated upon DEX+RUX treatment compared to the DNMT3A-wildtype group. Moreover, baseline BIRC5 expression is higher in DNMT3A-mutant T-ALL PDXs compared to the DNMT3A-wildtype group. Following treatment with the BIRC5 inhibitor YM155, the viability of DNMT3A-mutant T-ALL cells decrease significantly compared to DNMT3A-wildtype PDXs, which were relatively unaffected. In addition, we observed a similar reduction in cell viability in DNMT3A-mutant PDXs following BIRC5 siRNA nucleofection compared to the DNMT3A-wildtype group. Cumulatively, these studies reveal that enhanced JAK/STAT signaling promotes survival and chemoresistance in T-ALL patient cells with DNMT3A mutations and identify the JAK/STAT target gene BIRC5 as a specific genetic dependency of DNMT3A-mutant T-ALL cells. These data provide a critical first step towards a novel target for precision medicine approaches for this patient group.

Homoharringtonine Synergizes Strongly with Venetoclax Against the Early T-Cell Progenitor Acute Lymphoblastic Leukemia：from Bench to Bed

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with poor outcome and novel therapies are urgently needed. Venetoclax (VEN), a BCL2 inhibitor, has been approved by FDA for AML. Combination of VEN and demethylating drugs or targeted therapies such as MCL1 inhibitor showed promising effect in AML. Loucy, an ETP-ALL cell line, was also reported high expression of BCL2. But little is known about VEN combination treatment in ETP-ALL. Our data showed that homoharringtonine (HHT), a plant alkaloid, downregulated MCL1 in T-ALL. Herein, we examined the treatment effect of VEN combined with HHT on ETP-ALL using murine model and patient samples. First, we compared BCL2 levels and treatment effect of VEN alone on ETP-ALL (i.e., Loucy) and T-ALL (i.e., Jurkat, Molt4, and CCRF-CEM) cell lines. We observed higher levels of BCL2 and lower IC50 values (IC50 at 24h: 0.093 uM vs.>1uM) in Loucy vs T-ALL cells. Similar results were found in human samples, with IC50 of 0.539uM in ETP-ALL sample vs 3.165-23.25uM in T-ALL samples treated with VEN for 24h. Next, to determine whether the combination of VEN and HHT shows a synergistic effect on ETP-ALL, Loucy cells were treated with a serial concentration of VEN (range: 0.025-0.1uM) and HHT (range:10-40ng/mL) alone and in combination for 24h. VEN and HHT combination showed significant synergism in reducing cell viability, with a mean combination index (CI) of < 0.1. We observed similar results in patient samples, with mean CI lower than 0.01 indicating highly synergistic effect. To assess this in vivo, a cohort of ETP-ALL mice generated by transplanting Loucy cells into NSG mice were randomly divided into 4 groups and treated with vehicle, HHT (1 mg/kg, ip), VEN (25 mg/kg, oral gavage) or HHT+VEN combination. HHT and VEN combination significantly prolonged survival of the ETP-ALL mice compared to either drug alone or vehicle (median survival of combination vs VEN vs HHT vs control: 113.5 vs 95.5 vs 85.5 vs 69.0 days, p<0.01), suggesting a synergistic effect of HHT and VEN in vivo. To identify the mechanical basis for the synergy of VEN and HHT in ETP-ALL, we performed single-cell RNA-seq using primary patient ETP-ALL cells treated with vehicle, HHT, VEN or their combination for 24h. Based on the gene expression profile, we identified a cluster of ETP-ALL cells sensitive to HHT , and a cluster only sensitive to combination of VEN and HHT which showed higher levels of Bcl2 and Mcl1 and upregulated anti-apoptosis signal by gene set enrichment analysis (GSEA), suggesting intra-tumoral heterogeneity of ETP-ALL blasts. These results suggest that HHT or VEN alone is not sufficient to induce apoptosis in the resistant cells and combination of HHT and VEN could induce apoptosis by reducing MCL1 expression in these cells which was confirmed by western blot. Further, with the promising results from bench work and given that both HHT and VEN are approved anti-leukemia drugs, we translated these findings into clinic. The HAG (HHT, low dose Cytarabine, G-CSF priming) regimen has been widely used in China for the treatment of AML for 15 years and proved to be effective and safer than intensive chemotherapy. Based on above findings, with informed consent from patients, we applied venetoclax plus HAG(V-HAG) regimen to 3 relapsed/refractory(R/R) ETP-ALL patients and 4 newly diagnosed (ND) patients, including an 82-year-old refractory patient. CR/CRi rates after the first cycle treatment were 100%, which was significantly higher than the current induction therapy (CR rate ranging from 33.3-55.9% after the first induction cycle). Except for the 82-year-old patient who refused post-remission treatment, all the remaining 6 patients remained in remission until today. Of note, no clinical manifestations of tumor lysis syndrome were observed. The median time to absolute neutrophil count recovery of >500/μL and >1000/μL was 13 (range: 0-22) and 16 (range: 0-24) days, and to platelet count recovery of > 20,000/ μL was 16 (range: 0-21) days, indicating this regimen was relatively safe and well tolerated. In conclusion, our findings demonstrate that HHT and VEN showed synergistic effect on ETP-ALL and HHT/VEN based therapy is a promising and safe regimen in newly diagnosed and R/R ETP-ALL patients. With these promising results from bench to bedside, 2 multi-center phase Ib/II clinical trials using V-HAG in patients with R/R ETP-ALL and ND-ETP have been initiated at our institution.

GATA-3 is a proto-oncogene in T-cell lymphoproliferative neoplasms

Neoplasms originating from thymic T-cell progenitors and post-thymic mature T-cell subsets account for a minority of lymphoproliferative neoplasms. These T-cell derived neoplasms, while molecularly and genetically heterogeneous, exploit transcription factors and signaling pathways that are critically important in normal T-cell biology, including those implicated in antigen-, costimulatory-, and cytokine-receptor signaling. The transcription factor GATA-3 regulates the growth and proliferation of both immature and mature T cells and has recently been implicated in T-cell neoplasms, including the most common mature T-cell lymphoma observed in much of the Western world. Here we show that GATA-3 is a proto-oncogene across the spectrum of T-cell neoplasms, including those derived from T-cell progenitors and their mature progeny, and further define the transcriptional programs that are GATA-3 dependent, which include therapeutically targetable gene products. The discovery that p300-dependent acetylation regulates GATA-3 mediated transcription by attenuating DNA binding has novel therapeutic implications. As most patients afflicted with GATA-3 driven T-cell neoplasms will succumb to their disease within a few years of diagnosis, these findings suggest opportunities to improve outcomes for these patients.

T-Cell Progenitors As A New Immunotherapy to Bypass Hurdles of Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of preference for numerous malignant and non-malignant hemopathies. The outcome of this approach is significantly hampered by not only graft-versus-host disease (GvHD), but also infections and relapses that may occur because of persistent T-cell immunodeficiency following transplantation. Reconstitution of a functional T-cell repertoire can take more than 1 year. Thus, the major challenge in the management of allogeneic HSCT relies on the possibility of shortening the window of immune deficiency through the acceleration of T-cell recovery, with diverse, self-tolerant, and naïve T cells resulting from de novo thymopoiesis from the donor cells. In this context, adoptive transfer of cell populations that can give rise to mature T cells faster than HSCs while maintaining a safety profile compatible with clinical use is of major interest. In this review, we summarize current advances in the characterization of thymus seeding progenitors, and their ex vivo generated counterparts, T-cell progenitors. Transplantation of the latter has been identified as a worthwhile approach to shorten the period of immune deficiency in patients following allogeneic HSCT, and to fulfill the clinical objective of reducing morbimortality due to infections and relapses. We further discuss current opportunities for T-cell progenitor-based therapy manufacturing, including iPSC cell sources and off-the-shelf strategies. These opportunities will be analyzed in the light of results from ongoing clinical studies involving T-cell progenitors.

CD4+ progenitor cells sustain helper responses during chronic infection

How CD4+ Tcell responses are maintained during chronic infection is unknown. In this issue of Immunity, Xia etal. (2022) identify a progenitor Tcell subset that gives rise to effector and follicular helper Tcells to sustain antiviral responses.

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

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