T-cell Acute Lymphoblastic Leukemia Therapy Research Articles

Selective Inhibition of Deamidated Triosephosphate Isomerase by Disulfiram, Curcumin, and Sodium Dichloroacetate: Synergistic Therapeutic Strategies for T-Cell Acute Lymphoblastic Leukemia in Jurkat Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a challenging childhood cancer to treat, with limited therapeutic options and high relapse rates. This study explores deamidated triosephosphate isomerase (dTPI) as a novel therapeutic target. We hypothesized that selectively inhibiting dTPI could reduce T-ALL cell viability without affecting normal T lymphocytes. Computational modeling and recombinant enzyme assays revealed that disulfiram (DS) and curcumin (CU) selectively bind and inhibit dTPI activity without affecting the non-deamidated enzyme. At the cellular level, treatment with DS and CU significantly reduced Jurkat T-ALL cell viability and endogenous TPI enzymatic activity, with no effect on normal T lymphocytes, whereas the combination of sodium dichloroacetate (DCA) with DS or CU showed synergistic effects. Furthermore, we demonstrated that dTPI was present and accumulated only in Jurkat cells, confirming our hypothesis. Finally, flow cytometry confirmed apoptosis in Jurkat cells after treatment with DS and CU or their combination with DCA. These findings strongly suggest that targeting dTPI represents a promising and selective target for T-ALL therapy.

Abstract 5117: CK2 inhibiton exhibits synergistic effect in acute lymphoblastic leukemia

Abstract Survival for pre-B cell Acute lymphoblastic leukemia (B ALL) has improved greatly, however. High risk (HR) subgroups continue to result in significant mortality and morbidity of pediatric oncology patients. Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a lethal disease, often associated with high-risk clinical features. T-ALL therapy has made less improvement than B ALL and continues to represent a significant number of high-risk outcomes. Novel treatment strategies are required to overcome chemotherapy resistance and improve morbidity for HR B ALL. The development of treatments that can overcome chemotherapy resistance is essential for improving survival in T-ALL. Deletions of IKZF1, resulting in decreased IKAROS function, are present in High-risk ALL patients. Casein kinase 2 (CK2), a protein kinase upregulated in ALL, phosphorylates IKAROS preventing DNA binding reducing transcriptional regulation in ALL patients. Past studies have shown that pharmacologic inhibition of CK2 with CX-4945 rescues IKAROS function as a transcriptional regulator. Receptor tyrosine kinase c-KIT is transcriptionally regulated by IKAROS and is often upregulated in cancer. The purpose of this study was to determine whether restoration of IKAROS via CX-4945 would act in a synergistic manner with c-KIT inhibitor imatinib in ALL.Methods/results B-ALL Nalm6 cells were treated with imatinib over 72-hours to establish the half-maximal inhibitory concentration (IC50). The IC50 was used to determine the concentration range of imatinib to be used in combination with CX-4945 in a 72-hour synergy analysis of cytotoxicity.. Cell viability was measured using WST-1 cell proliferation assay. The data were analyzed using Calcusyn and synergy was defined as a combination index below 0.85. The study was repeated to determine a more precise range of synergy. The cytotoxicity of single drug c-KIT inhibitor, Imatinib, in Nalm6 B-ALL yielded an IC50 of 25uM while CK2 inhibitor CX-4945 resulted in 4uM. When a combination was administered with a constant dose of CX-4945 at 4uM and varying concentrations of imatinib, synergy was observed at multiple dose combinations. Conclusion: The data shows that CK2 inhibitor, CX-4945, and c-KIT inhibitor, imatinib, exhibit a synergistic therapeutic effect. Imatinib is indicated for use in Philadelphia positive (Ph+) acute lymphoblastic leukemia so future studies will include testing CX-4945/imatinib synergy in Ph+ BALL where we expect to see more potent synergistic effect. Citation Format: Daniel Gary Bogush, Joseph Schramm, Katarina Dovat, Yali Ding, Chingakham Singh, Sinisa Dovat. CK2 inhibiton exhibits synergistic effect in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5117.

CD99 tumor associated antigen is a potential target for antibody therapy of T-cell acute lymphoblastic leukemia.

Monoclonal antibodies (mAbs) are an effective drug for targeted immunotherapy in several cancer types. However, so far, no antibody has been successfully developed for certain types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). T-ALL is an aggressive hematologic malignancy. T-ALL patients who are treated with chemotherapeutic drugs frequently relapse and become drug resistant. Therefore, antibody-based therapy is promising for T-ALL treatment. To successfully develop an antibody-based therapy for T-ALL, antibodies that induce death in malignant T cells but not in nonmalignant T cells are required to avoid the induction of secondary T-cell immunodeficiency. In this review, CD99 tumor associated antigen, which is highly expressed on malignant T cells and lowly expressed on nonmalignant T cells, is proposed to be a potential target for antibody therapy of T-ALL. Since certain clones of anti-CD99 mAbs induce apoptosis only in malignant T cells, these anti-CD99 mAbs might be a promising antibody drug for the treatment of T-ALL with high efficiency and low adverse effects. Moreover, over the past 25 years, many clones of anti-CD99 mAbs have been studied for their direct effects on T-ALL. These outcomes are gathered here.

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

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

CD28 CAR T Cells for the Treatment of T Cell Malignancies

CONCLUSION While synthetic immunotherapy has been introduced into the standard of care treatment of patients with B-lineage malignancies, patients with T-lineage malignancies have not gained benefit from this novel approach, yet. The main reason is the challenging choice of target antigen in T-lineage malignancies such as pediatric T cell acute lymphoblastic leukemia (T-ALL). As activating mutations of CD28 have already been described in T cell malignancies, we set out to quantify surface expression of CD28 and other costimulatory molecules on primary pediatric T-ALL samples and explore the utility of this functionally relevant molecule as a novel target antigen. Bone marrow (BM) samples of pediatric T-ALL patients (n=54) at time of diagnosis and healthy control individuals (n=14) were collected. We quantified surface expression of CD28 and 25 additional co-stimulatory or co-inhibitory molecules by flow cytometry after gating on T cell precursors (living singlets, CD45 dim/SSC-A low and CD7 +). Subsequently, we generated a set of 20 CD28 directed second-generation chimeric antigen receptors (CARs) based on five different monoclonal antibodies with variation in hinge domain and light chain / heavy chain chronology. We analyzed specific CAR activity against CD28 expressing T-ALL cell lines after retroviral transduction into primary human T cells. Additional CD28 knockout (KO) by CRISPR/Cas9 could prevent T cell fratricide. CAR constructs were subjected to in vivo testing in a T-ALL NSG mouse model with transplantation of 7.5e4 CCRF-CEM cells transduced with firefly luciferase on day 0 and transfer of 2.5e6 CD28 KO T cells on day 3 harboring either of both CD28 CAR molecules. We used CD7 CAR T cells as positive control, CD19 CAR T cells and CD28 KO T cells without CAR as negative controls. We observed significant upregulation of CD28 on T-ALL leukemia when compared to healthy BM donors (Figure 1A, mean 68.8% vs. 3.7%, p=0.0002). We confirmed upregulation of CD28 in published T-ALL RNA-expression data sets. Interestingly, other co-stimulatory molecules such as CD127 were upregulated, too (mean 50.0% vs. 14.6%, p=0.0001), while co-inhibitory molecules such as CD160, TIGIT and TIM-3 were strongly downregulated (>10fold and p<0.0001 each). We hypothesized that this could imply a functional relevance of CD28 in T-ALL. Therefore, we performed co-culture assays of monocyte derived dendritic cells (CDs) expressing CD28 ligands CD80 and CD86 with different T-lineage leukemia cell lines. The presence of DCs significantly increased proliferation of CD28 + T-ALL cell lines under stress conditions. In order to test the feasibility of CD28 CAR T cells, we generated CD28 KO primary T cells without CAR. When tested in co-culture assays with leukemia cells and T cell engagers, CD28 KO T cells showed unchanged cytotoxic capacity and target-dependent activation illustrating that CD28 KO T cells retain short-term effector functions. Based on cytotoxic capacity and CAR T cell expansion, we identified two lead CD28 CAR candidates out of a pool of 20 CD28 CARs. After CD28 KO, CD28 CAR-T cells showed in vitro expansion comparable to CD19 CAR T cells. Next, we asked how CD28 CAR T cells compare to CD7 CAR T cells, which are currently evaluated clinical trials for T-ALL therapy. Therefore, we generated CD7 KO T cells that were subsequently transduced with a functional CD7 CAR. In vitro, both CD28 CAR T cells and CD7 CAR T cells showed killing of CD28 +CD7 + CCRF-CEM cells of >90% at an effector:target (E:T) ratio of 0.2:1, with CD7 being outperformed by one of the CD28 CARs while outperforming the other CD28 CAR at an E:T ratio of 0.04:1 (Figure 1B). Finally, we went on to validate the functionality of CD28 CAR T cells in vivo. We observed significantly prolonged survival of mice treated with CD28 CAR T cells and CD7 CAR T cells when compared to CD19 CAR T cells or control T cells. No difference in survival between CD7 and CD28 CAR T cell treated mice could be observed in two independent experiments. We identify CD28 as novel target antigen for pediatric T-ALL and provide evidence that CD28 is an immunotarget with functional relevance for T-ALL. We demonstrate the feasibility of CD28 CAR T cell generation and that these novel CAR T cells perform equally well as CD7 CAR T cells both in vitro and in vivo. Novel and functionally relevant CAR targets will facilitate clinical development of immunotherapy for T-lineage malignancies.

Single-Cell Transcriptomics Points to a Role of Stemness and Bone Marrow Niche Interactions in the Poor Response to Induction Therapy in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis with a 15-20% relapse rate. The induction therapy response is the strongest predictor of relapse and shows great variation between patients. However, whether intra-patient leukemic cell heterogeneity plays a role for the induction therapy response has not yet been investigated. We studied 13 patients with T-ALL (11 pediatric and 2 young adult, median follow-up 37 months). Of these, three were good responders (end of induction (EOI) minimal residual disease (MRD) undetectable/non-quantifiable by flowcytometry and PCR), while ten had a poor response (EOI MRD>0.01%, (range 0.05-97%). Of the ten poor responders, three experienced relapse and one died from resistant disease. Bone marrow samples from diagnosis (n=13) and paired samples from relapse (n=3) or MRD (n=4, MRD >5%) were subjected to combined single-cell RNA sequencing, single-cell surface marker profiling by antibody-derived tag (ADT)-sequencing, and single-cell TCR sequencing, using the 10X Genomics platform. A published healthy bone marrow dataset was used for cell annotation and clear distinction was observed between healthy cell subsets and leukemic blasts. TCR sequencing data revealed a hyperexpanded clone in six patients, with identical clonal sequences at diagnosis and relapse(n=2)/MRD(n=1). Surface marker profiling using a panel of 134 surface antigens showed good correspondence between leukemia-associated immunophenotypes found by ADT-sequencing and by flow cytometry for 16 common clinically used markers. To examine factors associated with a poor therapy response, we compared average gene expression of leukemic cells in poor and good responders at diagnosis using differential gene expression (DGE) and gene set enrichment analysis (GSEA). At diagnosis, poor responders showed upregulation of genes involved in extracellular matrix (ECM) degradation/organization, including the adhesion molecules ITGA5 and PECAM1, and MMP11, while showing downregulation of cell cycle genes. Accordingly, the fraction of cells predicted to be in G0/G1 phase at diagnosis of poor responders positively correlated with EOI MRD (r=0.52, p=0.07, Spearman's rank correlation). To investigate if this high fraction of quiescent cells in poor responders was associated with a more stem-like cell state, we examined the expression of a published hematopoietic stem cell (HSC) signature (Forsberg et al, 2010) that we previously selected for investigation in BCP-ALL (Modvig et al, Molecular Oncology, 2022). Indeed, patients with a poor response had a higher percentage of leukemic blasts with an HSC signature module score above zero (p=0.028, Kolmogorov-Smirnov, distribution in Figure 1), suggestive of stemness as a contributing factor to therapy resistance. To define molecular changes in leukemic blasts during therapy, we compared the leukemic blast gene expression profiles in matched diagnostic and MRD samples using DGE and GSEA. This revealed that the leukemic blasts within the first month of therapy downregulated cell cycle genes, while upregulating chemokine receptors, such as CXCR4. To identify specific genes associated with high MRD, we combined genes upregulated in poor responders (n=314, p<0.01, DGE) with genes associated with EOI MRD levels (n=98, r>0.8 and p<0.001, rank regression). This resulted in a 30-gene signature, comprising several genes previously associated with cancer stem cells, such as MICAL3, ELF4, and NLRP3, and genes encoding proteins involved in cancer drug resistance. These included sialyl transferases, acid ceramidase ( ASAH1) and zyxin ( ZYX). ZYX silencing was previously shown to downregulate the anti-apoptotic protein BCL-2 in leukemia. Accordingly, the percentage of cells with detectable BCL2 expression at diagnosis correlated with EOI MRD (r=0.58, p=0.04), supporting a role of BCL-2 inhibitor therapy in T-ALL. In conclusion, our data suggest that therapy-resistant cells are more stem-like and quiescent at diagnosis, display upregulation of genes involved in bone marrow niche ECM organization and adhesion, and genes related to drug resistance. Likewise, residual leukemic blast following therapy show less proliferation and CXCR4 upregulation. Thus, single-cell transcriptomics provides important molecular insights into therapy-resistant leukemic cells and could comprise a valuable prognostic tool in T-ALL.

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and thought to induce transformation in part through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

CD38-Specific CAR Integrated into CD38 Locus Driven by Different Promoters Causes Distinct Antitumor Activities of T and NK Cells.

The robust and stable expression of CD38 in T-cell acute lymphoblastic leukemia (T-ALL) blasts makes CD38 chimeric antigen receptor (CAR)-T/natural killer (NK) a potential therapy for T-ALL. However, CD38 expression in normal T/NK cells causes fratricide of CD38 CAR-T/NK cells. Here a "2-in-1" gene editing strategy is developed to generate fratricide-resistant locus-specific CAR-T/NK cells. CD38-specific CAR is integrated into the disrupted CD38 locus by CRISPR/Cas9, and CAR is placed under the control of either endogenous CD38 promoter (CD38KO/KI ) or exogenous EF1α promoter (CD38KO/KI EF1α). CD38 knockout reduces fratricide and allows the expansion of CAR-T cells. Meanwhile, CD38KO/KI EF1α results in higher CAR expression than CD38KO/KI in both CAR-T and CAR-NK cells. In a mouse T-ALL model, CD38KO/KI EF1α CAR-T cells eradicate tumors better than CD38KO/KI CAR-T cells. Surprisingly, CD38KO/KI CAR-NK cells show superior tumor control than CD38KO/KI EF1α CAR-NK cells. Further investigation reveals that endogenous regulatory elements in NK cells lead to higher expression of CD38 CAR than in T cells, and the expression levels of CAR affect the therapeutic outcome of CAR-T and CAR-NK cells differently. Therefore, these results support the efficacy of CD38 CAR-T/NK against T-ALL and demonstrate that the "2-in-1" strategy can resolve fratricide and enhance tumor eradication, paving the way for clinical translation.

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

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

Allogenic and autologous anti-CD7 CAR-T cell therapies in relapsed or refractory T-cell malignancies

Chimeric antigen receptor-T (CAR-T) therapy remains to be investigated in T-cell malignancies. CD7 is an ideal target for T-cell malignancies but is also expressed on normal T cells, which may cause CAR-T cell fratricide. Donor-derived anti-CD7 CAR-T cells using endoplasmic reticulum retention have shown efficacy in patients with T-cell acute lymphoblastic leukemia (ALL). Here we launched a phase I trial to explore differences between autologous and allogeneic anti-CD7 CAR-T therapies in T-cell ALL and lymphoma. Ten patients were treated and 5 received autologous CAR-T therapies. No dose-limiting toxicity or neurotoxicity was observed. Grade 1–2 cytokine release syndrome occurred in 7 patients, and grade 3 in 1 patient. Grade 1–2 graft-versus-host diseases were observed in 2 patients. Seven patients had bone marrow infiltration, and 100% of them achieved complete remission with negative minimal residual disease within one month. Two-fifths of patients achieved extramedullary or extranodular remission. The median follow-up was 6 (range, 2.7–14) months and bridging transplantation was not administrated. Patients treated with allogeneic CAR-T cells had higher remission rate, less recurrence and more durable CAR-T survival than those receiving autologous products. Allogeneic CAR-T cells appeared to be a better option for patients with T-cell malignancies.

Synthetic oleanane triterpenoids suppress MYB oncogene activity and sensitize T-cell acute lymphoblastic leukemia cells to chemotherapy.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with poor prognosis. The MYB oncogene encodes a master transcription factor that is activated in the majority of human T-ALLs. In the present study, we have performed a large-scale screening with small-molecule drugs to find clinically useful inhibitors of MYB gene expression in T-ALL. We identified several pharmacological agents that potentially could be used to treat MYB-driven malignancies. In particular, treatment with the synthetic oleanane triterpenoids (OTs) bardoxolone methyl and omaveloxolone decreased MYB gene activity and expression of MYB downstream target genes in T-ALL cells with constitutive MYB gene activation. Notably, treatment with bardoxolone methyl and omaveloxolone led to a dose-dependent reduction in cell viability and induction of apoptosis at low nanomolar concentrations. In contrast, normal bone marrow-derived cells were unaffected at these concentrations. Bardoxolone methyl and omaveloxolone treatment downregulated the expression of DNA repair genes and sensitized T-ALL cells to doxorubicin, a drug that is part of the standard therapy of T-ALL. OT treatment may thus potentiate DNA-damaging chemotherapy through attenuation of DNA repair. Taken together, our results indicate that synthetic OTs may be useful in the treatment of T-ALL and potentially also in other MYB-driven malignancies.

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

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

Chimeric antigen receptor T-cell therapy for T-ALL and AML.

Non-B-cell acute leukemia is a term that encompasses T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Currently, the therapeutic effectiveness of existing treatments for refractory or relapsed (R/R) non-B-cell acute leukemia is limited. In such situations, chimeric antigen receptor (CAR)-T cell therapy may be a promising approach to treat non-B-cell acute leukemia, given its promising results in B-cell acute lymphoblastic leukemia (B-ALL). Nevertheless, fratricide, malignant contamination, T cell aplasia for T-ALL, and specific antigen selection and complex microenvironment for AML remain significant challenges in the implementation of CAR-T therapy for T-ALL and AML patients in the clinic. Therefore, designs of CAR-T cells targeting CD5 and CD7 for T-ALL and CD123, CD33, and CLL1 for AML show promising efficacy and safety profiles in clinical trials. In this review, we summarize the characteristics of non-B-cell acute leukemia, the development of CARs, the CAR targets, and their efficacy for treating non-B-cell acute leukemia.

Homoharringtonine Exhibits Anti-Tumor Effect in T-Cell Acute Lymphoblastic Leukemia By Targeting Notch1/Myc Pathway

T cell acute lymphoblastic leukemia (T-ALL) is a highly proliferative hematologic malignancy, mainly mediated by NOTCH/MYC signaling. Therefore, new therapies targeting NOTCH/MYC pathway are urgently required. Homoharringtonine (HHT), a potent inhibitor of translation elongation, has been used in China for many years for the treatment of myeloid leukemia and was approved in 2012 by the US Food and Drug Administration for the treatment of chronic myeloid leukemia with resistance to tyrosine kinase inhibitors. However, little is known about its efficacy in T-ALL. Herein, we examined the therapeutic role of HHT in T-ALL using mouse model and patient cells. First, we treated T-ALL cell lines (i.e., Jurkat, Molt4 and CCRF-CEM) and patient samples with HHT in vitro. After 24-hour(h) treatment, we observed very low IC50 values (5-20ng/mL) in all three T-ALL cell lines, suggesting highly sensitive to HHT treatment. HHT dramatically induced apoptosis and cell cycle arrest in G0/G1 phase in all three T-ALL cell lines. We observed similar results in human T-ALL cells treated with HHT for 24h, with much lower IC50 values (2-5ng/mL vs 30-110 ng/mL) in all three T-ALL patient samples compared to mononuclear cells from healthy donors, with increased apoptosis rates (89.2% vs 29.6%, p<0.001). Collectively, these results showed that T-ALL cell lines and patient samples exhibit high sensitivity to HHT in vitro. To evaluate the in vivo efficacy of HHT in T-ALL, we established a cohort of T-ALL mice by transplanting Luci+ Jurkat cells into NSG mice and then treated these mice with HHT (1mg/kg, ip) or vehicle (PBS) for 10 days, starting on day 9 post transplantation. Leukemia burden was compared by measuring Bioluminescence and engraftment at day 14 and day 19 post transplantation in HTT-treated and PBS-treated groups. We observed significantly decreased leukemia burden in HHT-treated mice compared to PBS-treated controls (median total flux: 796.3 vs 3701*106 at day 19, P=0.0001). HHT significantly prolonged the survival of the T-ALL mice as compared to PBS treatment (median survival: 29 vs 25 days, P=0.0003). T-ALL patient-derived-xenograft (PDX) model was also constructed and treated with HHT or PBS for 10 days. We observed significantly reduced human T-ALL engraftment rates (14.4-25.3% vs 37.8-65.3% at day 10 post transplantation, p<0.0001) and prolonged survival (median survival: 54 vs 45 days, P=0.0098) in the HHT-treated PDX compared to PBS-treated PDX, suggesting an in vivo anti-leukemia effect of HHT in T-ALL. Next, we performed RNA-seq of T-ALL cells with or without HHT treatment to identify downstream pathways that are regulated by HHT. Through gene set enrichment analysis (GSEA), we found that Myc pathway and Notch pathway were among the top 6 upregulated gene sets. Further, using western-blot analysis, we showed that all the expression levels of Notch1, c-MYC and MCL-1 were decreasing in T-ALL cell lines and patient samples after exposing to increased concentrations of HHT, suggesting that they were all down-regulated by HHT. Interestingly, we observed that a much lower concentration of HHT is needed to down-regulate Notch 1 (4ng/mL) than that needed to down-regulate MCL1(8-16ng/mL). Similar results were found in T-ALL human samples. Taken together, these results suggest that HHT could attenuate NOTCH 1 and MYC expression in T-ALL cells. Further, we used the Notch-1 induced T-ALL mouse model to verify the effect of HHT on Notch pathway and test its in vivo anti-ALL efficacy. Strikingly, we found that HHT treatment significantly reduced white blood cell (WBC) counts (0.82-1.68 *109/L vs 9.73-17.46*109/L, p<0.0001) and T-ALL burden (0.0-0.8% vs 7.9-57.1%, p=0.0003) and significantly prolonged survival (median survival: not reached vs 31 days, p=0.0007) as compared to PBS treatment. Interestingly, 60% (six of ten) HHT-treated T-ALL mice versus 0% of PBS-treated mice, obtained long-term survival, free of T-ALL after monitoring for 75 days, suggesting that these mice likely are cured by HHT monotherapy. QPCR and western-blot analysis using bone marrow cells of the treated mice showed that HHT dramatically reduced both the mRNA and protein levels of NOTCH1 compared to the control group. In summary, we showed here that HHT, approved by the FDA for CML treatment, exhibited in vivo therapeutic efficacy in T-ALL, by inhibiting the NOTCH-MYC pathway. Our data support to explore HHT as a novel therapy for T-ALL in the clinic.

PDGFR-β As a New Therapeutic Target in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. General prognosis of T-ALL has been improving over time. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains dismal. PDGFR-β is a transmembrane glycoprotein dimer molecule that functions as a receptor tyrosine kinase. Ligand binding (PDGF-B/PDGF-D) results in receptor dimerization and subsequent activation via autophosphorylation, causing the downstream activation of various signaling pathways such as PI3K/AKT, MAPK/ERK, JAK/STAT and Notch. During normal T cell development, PDGFR-β is expressed in the double negative stage in CD34+ T-cells and drops dramatically once CD34 is lost. The expression of both ligands, PDGF-B and PDGF-D, seems to be restricted to γδ CD3+ CD1- T-cells. We looked in vivo to the effect of PDGFR-β overexpression on normal T cell development making use of CD2-iCre driven overexpression of PDGFR- β in a C57Bl6 background. Overexpression of PDGFR-β in the R26-locus did not affect normal T-cell development. In T-ALL patients, high PDGFR-β expression can be observed in the HOXA and TLX3 subgroups. These subgroups have significantly higher PDGFR-β expression compared to normal developing T-cells. As constitutively active PDGFR-β is observed in a variety of malignancies, multi-target kinase inhibitors are already used in the clinic. However, they are not specific and often result in adverse side-effects. CP-673451 is a novel, potent and selective PDGFR inhibitor. Mechanistically, CP-673451 inhibits autophosphorylation of dimeric PDGFR-β and in this way prevents phosphorylation of downstream PI3K/AKT, GSK-3α and GSK-3β. Previous studies showed growth inhibition of multiple tumor xenografts (lung and colon carcinomas, glioblastoma) mainly due to a direct antitumor effect. Previously, we demonstrated the efficacy of CP-673451 on a T-ALL (IC50=544 nM) and a B-ALL cell line (IC50=500nM) with high PDGFR-β expression, as well as on a T-ALL PDX model with high levels of PDGFR-β (IC50=1,6 nM). Both cell lines and PDX models showed phosphorylation of the PDGFR-β receptor. After PDGFR-β inhibition, a decrease of PDGFR-β could be observed as a negative feedback loop. Also, phosphorylation of the receptor itself decreased which resulted downstream in a downregulated phosphorylation levels of GSK3β (Ser9), STAT3 (Tyr705) and STAT5 (Tyr694). Combination experiments with glucocorticoids (Fig. A) and chemotherapy (Fig. B), currently used as conventional therapy in T-ALL, showed a synergistic effect when combined with PDGFR-β inhibition. In conclusion, we have shown that specific PDGFR-β inhibitors can be used as a novel and targeted therapy in PDGFR-βhigh T-ALL patients and that they can be of added value to the current chemotherapy treatment for T-ALL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Synergistic Sensitivity of Selinexor with Chidamide By Targeting NF-Κb /c-FLIP Signaling Via Suppressing HDAC1/XPO1 Activity in T-ALL

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor prognosis. Exportin-1 (XPO1) overexpression is observed in ALL and is associated with a poor prognosis for the patients. Recently, XPO1 inhibitor is reported to show anti-tumor effects in T-ALL. HDAC inhibitors (HDACi) are also reported to inhibit T-cell leukemia, and Chidamide is a new selective HDACi. Here, we explored the effect of Chidamide combined with the XPO1 inhibitor Selinexor in T-ALL and the underlying mechanisms. Methods Cell Counting Kit-8 (CCK-8) was used for cell viability assay, Annexin-V/PI staining following flow cytometry analysis for apoptosis, and qPCR and Western Blot for gene expression in CEM and Molt4 T-ALL cell lines. CalcuSyn analysis was performed for a synergistic effect. Cellular fractionation combined with western blot was used to detect the distribution of observed proteins. The co-immunoprecipitation(Co-IP) assay was utilized for the protein-protein interaction. Results We first observed that both Chidamide and Selinexor induce cell proliferation arrest of T-ALL cells in a dose-dependent manner. Then we found that Selinexor significantly increased the Chidamide-mediated cell proliferation arrest in both CEM and Molt4 cells and CalcuSyn analysis showed they have a synergistic effect (Fig. 1A). Moreover, the combination significantly increased the apoptosis (Fig.1B) and cell cycle arrest at G0/G1 phase in CEM cells (Fig. IC) and Molt-4 cells compared to the single drug controls. Consistently, the combination significantly up-regulated the expression of pro-apoptotic factors Cleaved caspase-3 and BAX, but down-regulated that of anti-apoptotic effectors BcL2 and MCL1, and the cell cycle regulators CDK2 and CyclinE2 compared to that of the single drug controls in the cells (Fig.1D). These data together indicate the combination of Chidamide with Selinexor has synergistic anti-leukemia effects in T-ALL. To understand the underlying mechanism of the synergy, we observed the apoptosis regulatory proteins Cellular FLICE inhibitory protein(c-FLIP) is significantly down-regulated upon the combination treatment compared to the single drug controls (Fig. 1E). c-FLIP is reported to be transcriptionally regulated by NF-kB. Interestingly, we observed that Chidamide significantly down-regulates the expression of HDAC1 and NF-κB p50 in T-ALL cells (Fig. 1F). Moreover, IκB-α and NF-κB p65 are XPO1 cargoes, and the interaction of IκB-α with NF-κB p65 prevents NF-κB binding to target DNA. We also observed the accumulation of IκB-α, and NF-κB p65 in the nuclear fraction in CEM (Fig. 1G) and Molt4 cells upon the Selinexor treatment. The co-IP assay showed the IκB-α bound to NF-κB p65 subunit in the cells and this interaction is enhanced in nuclear faction upon Selinexor treatment (Fig.1H), which might prevent NF-κB dependent transcription of target genes such as c-FLIP. Furthermore, the combination of Selinexor with Chidamide showed significant effects on cell growth arrest, the expression of CDK2, CyclinE2, Cleaved caspase3, and BCL2 in primary cells from T-ALL patients (Fig.1I). Finally, qPCR results revealed c-FLIP was overexpressed in T-ALL patients compared to the normal controls in our T-ALL patient cohort (Fig.1J). These data together indicated that the combination exerts its anti-tumor effect by targeting XPO1/HDAC1/NF-kB/c-FLIP signaling. The mechanism model is shown in Fig.1K. Conclusions Our results indicated that the combination of Selinexor with Chidamide has synergistic sensitivity on cell proliferation arrest, apoptosis, and cell cycle arrest in T-ALL by targeting NF-kB/c-FLIP oncogenic signaling through suppressing XPO1/HDAC1 activity. Our data also provide experimental evidence for the further clinical trial of the new potential combination in therapy of T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Idasanutlin and Navitoclax Induce Synergistic Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

We report here investigation of the combination of idasanutlin and navitoclax in the context of T-cell acute lymphoblastic leukemia (T-ALL), including detailed mechanistic characterization and in vivo patient derived xenograft results. T-ALL is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. An unmet clinical need remains for the development of better therapies for T-ALL. Notch pathway activation mutations are present in the majority of T-ALL and are thought to induce transformation, in part, through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, and critical to our approach, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these genetic data, we hypothesized that treatment with MDM2 inhibitors, by bypassing the loss of ARF, would increase p53 expression and lead to T-ALL cell apoptosis. To test this hypothesis, we initially treated T-ALL patient derived xenograft (PDX) lines in vitro with single-agent idasanutlin (a second generation MDM2 inhibitor) and observed upregulation of pro-apoptotic p53 target genes Bax and Puma1. However, only modest T-ALL cell apoptosis was observed in response to MDM2 inhibition alone. Based on these observations, particularly the induction of pro-apoptotic p53 targets, we hypothesized that inhibition of pro-survival BH3 domain proteins would synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with navitoclax (ABT-263), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient and dose-limiting thrombocytopenia was observed. Combination therapy with idasanutlin could permit on-target inhibition with tolerable dosing. In vitro treatment of a panel of 5 different PDX lines derived from pediatric and adult T-ALL with dual idasanutlin and navitoclax led to robust cell death in all lines. In addition, treatment of a T-ALL PDX line with a matrix of dose combinations showed strong evidence of synergic activity. Synergy was assessed by ZIP (zero interaction potency) score, a statistical method developed by B Yadav et al. (Comput. Struct. Biotechnol. J. 2015) that compares the change in potency of individual dose response curves in the presence of a second agent. Generally, a ZIP score greater than 5 is considered significant; Idasanutlin and navitoclax combination therapy had a global ZIP score=16-18, with the most synergic dose area ZIP score=40-52. Combined treatment resulted in marked apoptosis, with strong induction of pro-apoptotic p53 targets, including PUMA and BAX. Finally, we assessed the in vivo response of four different T-ALL xenografts to dual treatment. In each case, human T-ALL cells were injected into immunodeficient NSG mice and randomized into treatment groups. Monotherapy with idasanutlin or navitoclax resulted in a modest but significant decrease in T-ALL burden in all cases, with the exception of an ETP T-ALL, which was resistant to idasanutlin monotherapy. A marked response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts. Specifically, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change in tumor burden for synergy interaction. Furthermore, overall survival was significantly increased in the combination treatment group. These data suggest that the rational combination of idasanutlin and navitoclax is highly active, with induction of an apoptotic cell death in T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Progenitor Sub-Populations in Treatment Resistant T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid malignancy for which optimal therapeutic approaches for relapsed/refractory disease and biomarkers for upfront risk stratification are currently unknown. Comprehensive, large-cohort bulk-level profiling of T-ALL is currently underway with the promise to reveal novel drivers and associate genetics with treatment response. To synergize with these studies, we designed a single-cell (sc) genomics study powered to comprehensively characterize intra-tumoral arrest states in T-ALL. Using integrated scRNA and scATAC-seq analysis on 40 T-ALL cases alongside a novel, single cell atlas of pediatric T-cell development, we identified and characterized a bone-marrow progenitor-like (BMP-like) tumor sub-population associated with resistance to conventional therapy across distinct immunophenotypic subtypes of T-ALL. We first compared 10 treatment-refractory ETP-ALL patients (End of Induction MRD > 20%) to 10 treatment sensitive ETP-ALL patients (EOI MRD negative), where we identified a strong and consistent enrichment of ETP-ALL blasts that were transcriptomically and epigenetically analogous to multipotent BMP. BMP-like blasts had higher surface protein expression of myeloid and stem cell markers, such as CD33, CD123, HLA-DR, and CD34, and lowered expression of T-lineage surface protein markers, such as sCD3, CD4, CD2, and CD10. The top differentially expressed (DE) genes for BMP-like blasts included stem cell markers (C1QTNF4, CD44, LGALS1, HOPX), myeloid-lineage (S100A4, SPINK2), and B-lineage markers (IGLL1, IGKC, IGHM). The top DE transcription factors (TFs) in BMP-like blasts included TFs associated with self-renewal (MEF2C, HOXA3/4/5/6/9/10/11, MEIS1, HHEX, SPI1) and recovery from genotoxic stress (BCL11A). Several of the genes involved in self renewal (MEF2C, HOXA9, FLI1), or T-cell developmental block (SPI1) also had increased motif accessibility. We integrated these signatures with large-cohort bulk-genomic results of 1300+ T-ALL patients treated in the Children's Oncology Group (COG) AALL0434 clinical trial. The molecular signature of BMP-like blasts predicted poor outcome across multiple subtypes of T-ALL (ETP & Non-ETP) and were validated in two independent COG trials (AALL0434, full cohort sequenced; AALL1231, partially sequenced cohort) with corresponding bulk-RNAseq data (Panel A, ETP-ALL patient stratification). We also utilized single-cell derived transcriptional signatures in conjunction with bulk-sample-derived mutation calls to identify fusion drivers (eg, MLLT10, KMT2A, NUP98, NUP214) and gene mutations (eg, NOTCH1 WT, IL7R WT, ETV6 mut, and SAT1B mut) that are associated with the BMP-like state and poor outcome. Computational analysis and in vitro experiments indicated that BMP-like tumors would be highly resistant to conventional ALL-directed cytotoxics. To address the pressing need for targeted therapy in chemo-refractory T-ALL, we performed multi-database drug screening against the BMP-like signature derived from our single cell data, nominating a number of surface markers (CD44, ITGA4, LGALS1) and intracellular targets (BCL-2, MCL-1, BTK, NFKB) for experimental follow-up in patient derived xenografts (PDX) which we generated and characterized in our single cell study. Notably, experimental studies of PDX expanded blasts indicated consistent and selective vulnerability of BMP-like blasts to apoptosis-inducing agents (Panel B). Other agents with potent activity in this population included TEC-kinase inhibitors and proteasome inhibitors. Follow-up in-vivo studies in PDX are underway with the promise to bridge these therapies to the clinic. Our work comprehensively defines the developmental arrest states of T-ALL in reference to human early T-cell development. By doing so, we reveal a previously unappreciated sub-population level overlap between patients with distinct subtypes of T-ALL and identify one common BMP-like sub-population that associates with poor response to current therapy. As T-ALL thus far is solely defined by bulk-sample immuno-phenotype, we propose the prospective identification of BMP-like sub-populations and use of non-conventional cytotoxic agents for upfront-risk-stratification and targeted therapy to be realized in high-risk T-ALL. JX & CC, as well as DTT & KT, contributed equally to the work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ALL-017 Adult T-Cell Acute Lymphoblastic Leukemia With Transducin-Like Enhancer Gene Expression

Transducin-like enhancer of split 1 (TLE1) is a member of the TLE family of transcriptional co-repressors that control the transcription of a wide range of genes. The aim of this study was to evaluate the prognostic role of TLE1 gene expression in patients with T-cell acute lymphoblastic leukemia (T-ALL). This study was conducted on 97 newly diagnosed T-ALL patients admitted to the Mansoura University oncology center (59 males and 38 females) with median age 33 years, in addition to 102 apparently healthy individuals who served as a control group. TLE1 gene expression was measured in both patients and control groups by real time - PCR. The calculation of relative gene expression was done using the AACt method. TEL1 gene expression was significantly down regulated in T-ALL cases (median 2.83) as compared to controls (median 84.65) (p < 0.001). Low TEL1 gene expression was significantly associated with CNS infiltration, failure to achieve 1st complete remission and higher relapse rate (p< 0.001, 0.001 and 0.023, respectively). Early T-cell precursor (ETP)-ALL/LBL subtype was found to be significantly associated with low TEL1 gene expression (P 0.04), however there was no relation between TEL1 gene expression and TCR rearrangement or other available molecular abnormalities. Likewise, low TEL1 gene expression was significantly associated with shorter overall survival (OS) and disease-free survival (DFS) (P= 0.012 and 0.011, respectively). Furthermore, Low TEL1 gene expression was considered a risk predictor of relapse with OR 3.636(CI.1.422-9.295) (P =0.007); and OR 0.803(CI. 0.609-0.96) (P=0.021) and an independent predictor of T-ALL patients' outcomes with OR 0.619 (CI. 0.44-0.872) (P=0.006). TLE1 gene expression was significantly down regulated in T-ALL as compared with controls. Low TLE1 expression is an independent predictor of the T-ALL patient's outcome and could open the way for further risk stratification and targeted therapy in T-ALL.

RHEB is a potential therapeutic target in T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.