T-cell Acute Lymphoblastic Leukemia Cell Research Articles

Calcicoptosis Induced by Purple Sweet Potato Anthocyanins through the Nonosmotic Regulation of the NFAT5/S100A4-S100A9 Pathway in Acute Lymphoblastic Leukemia.

Purple sweet potato is considered an abundant, inexpensive, and ideal source of anthocyanins. Purple sweet potato anthocyanins (PSPAs) have been shown to possess high antimutagenicity and antitumor effects due to the abundance of acylated anthocyanins. However, the effect and underlying mechanism of PSPA effects in acute lymphoblastic leukemia (ALL), especially T-cell acute lymphoblastic leukemia (T-ALL), remain unclear. In this study, the antileukemic effects of PSPAs and the underlying molecular mechanisms were evaluated by in vitro and in silico assays. PSPAs extracted from ten cultivars were analyzed and quantified. Anthocyanins from Nanzi 018, which showed the best antileukemic effect, were selected to analyze the underlying mechanism. First, the PSPAs potently reduced cell viability and induced apoptosis. Additionally, the PSPAs sharply increased intracellular Ca2+ levels, which resulted in calcium overload in T-ALL cells. Furthermore, on the basis of bioinformatics analyses, we focused on an osmotically regulated transcription factor, NFAT5. Molecular docking preliminarily indicated that PSPA molecules bound and interacted with the NFAT5 protein. Western blot analyses confirmed that PSPAs elicited calcium overload by nonosmotic regulation of NFAT5/S100A4-S100A9 pathway activation. Moreover, pretreatment with a NFAT5 inducer confirmed that PSPAs targeted NFAT5 and affected p38/NF-κB/Bcl-2/Caspase-3 axis activation. This study demonstrates that PSPAs exert their antileukemic effects through calcicoptosis induction by targeting NFAT5.

KLF9 promotes autophagy and apoptosis in T-cell acute lymphoblastic leukemia cells by inhibiting AKT/mTOR signaling pathway

KLF9 promotes autophagy and apoptosis in T-cell acute lymphoblastic leukemia cells by inhibiting AKT/mTOR signaling pathway

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

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

Effect of Netrin-1 on VEGFA Expression in T-ALL Cells and Its Related Mechanism

ELISA assays were applied to detect the levels of Netrin-1 and VEGFA in the bone marrow (BM) samples from children in the T-ALL and control group. The level of Netrin-1 and VEGFA were compared between control children and patients, and the liner correlation between Netrin-1 and VEGFA was analyzed. The T-ALL cells Jurkat and Molt-4 were culture in vitro, and the cells were treated with different concentration of Netrin-1 (0, 25, 50, 100 ng/ml) for 24 h, quantitative RT-PCR (qRT-PCR) and Western blot were used to detect the VEGFA expression in Jurkat, Molt-4 cells. The expression of Netrin-1 receptors in T-ALL cells was detected by qRT-PCR and the interaction between Netrin-1 and receptor in each cells was detected by co-IP. Furthermore, Western blot was used to detect the phosphorylation level of key prateins of AKT signal transduction pathway including Akt and mTOR in T-ALL cells treated with Netrin-1 (100 ng/ml). The expression of VEGFA and phosphorylation of AKT pathway transducers were detected by Western blot, after T-ALL cells treated with Netrin-1 (100 ng/ml) combined with inhibitors specific to Akt or mTOR. The expression level of Netrin-1 and VEGFA in T-ALL patients BM samples were both signi-ficantly higher than that of control group. And the expression level of Netrin-1 was positively correlated with that of VEGFA（r2=0974). With the increase of Netrin-1 concentration, the expression level of VEGFA also increased(P<0.05）. Netrin-1 interacted with its receptor, integrin-β4 at the Netrin-1 concentration of 100 ng/ml. Further, the treatment of Netrin-1 could increase the phosphorylation of Akt and mTOR, which were the key transducers of AKT pathway. After treatment of T-ALL cells with Netrin-1 (100 ng/mL) and Akt inhibitor, the expression of VEGFA and phosphorylation of Akt or mTOR decreased. When the cells were treated with Netrin-1(100 ng/ml) and mTOR inbititor, the phosphorylation level of mTOR and the expression of VEGFA decreased, the phosphorylation level of Akt increased. The expression of Netrin-1 and VEGFA in bone marrow of childred with T-ALL were abnormal, and there was a linear relationship between them. Netrin-1 can interact with its receptor, integrin-β4 and activate AKT transduction pathway to elevate the expression of VEGFA in T-ALL cells.

Inhibition of the m6A reader IGF2BP2 as a strategy against T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant leukemia with extremely limited treatment for relapsed patients. N6‐methyladenosine (m6A) reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) participates in the initiation and growth of cancers by communicating with various targets. Here, we found IGF2BP2 was highly expressed in T-ALL. Gain and loss of IGF2BP2 demonstrated IGF2BP2 was essential for T-ALL cell proliferation in vitro and loss of IGF2BP2 prolonged animal survival in a human T-ALL xenograft model. Mechanistically, IGF2BP2 directly bound to T-ALL oncogene NOTCH1 via an m6A dependent manner. Furthermore, we identified a small-molecule IGF2BP2 inhibitor JX5 and treatment of T-ALL with JX5 showed similar functions as knockdown of IGF2BP2. These findings not only shed light on the role of IGF2BP2 in T-ALL, but also provide an alternative γ‑Secretase inhibitors (GSI) therapy to treat T-ALL.

5-Aza-dC promotes T-cell acute lymphoblastic leukemia cell invasion via downregulation of DNMT1 and upregulation of MMP-2 and MMP-9

5-Aza-2'-deoxycytidine (5-Aza-dC) is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells, and can restore the expression of their target genes in Jurkat cells. The goal of this study was to discern the potential effect of 5-Aza-dC on the invasion of T-ALL cells in acute lymphoblastic leukemia (ALL). The role of matrix metallopeptidase (MMP)-2, MMP-9, and DNMT1 in cell invasion was determined using loss- and gain-of-function investigations in Jurkat- and Sup-T1-R cells. A nude mouse model of ALL was established for further exploration of their roles in vivo. MMP-2 and MMP-9 exhibited high expression and low DNA methylation levels in 5-Aza-dC-resistant T-ALL cells. DNMT1 was poorly expressed in 5-Aza-dC-resistant T-ALL cells and exhibited decreased enrichment in the promoter region of MMP-2 and MMP-9. Silencing of MMP-2 and MMP-9 or DNMT1 overexpression reduced T-ALL cell invasion. After treatment of Sup-T1 cells with 5-Aza-dC, MMP-2 and MMP-9 presented with reduced DNA methylation levels but increased expression, and DNMT1 expression was identified to be suppressed. Further, in vivo assays revealed that DNMT1 alleviated T-ALL by reducing the expression of MMP-2 and MMP-9 in vivo. All in all, 5-Aza-dC activates MMP-2 and MMP-9 expression by reducing DNMT1-dependent DNA methylation levels and, hence, promotes the invasion of T-ALL cells.

The characteristics and prognostic significance of the SET-CAN/NUP214 fusion gene in hematological malignancies: A systematic review.

Background:The SET-CAN/NUP214 fusion gene resulting from chromosomal del(9)(q34.11q34.13) or t(9;9) (q34;q34) has been found in T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML) and myeloid sarcoma (MS). Furthermore, the SET-CAN/NUP214 fusion gene has been found in the T-ALL cell line LOUCY and the AML line MEGAL. The common features of these cases are insensitivity to chemotherapy and poor prognosis. We reviewed the characteristics and prognostic significance of the SET-CAN/NUP214 fusion gene in hematological malignancies.Methods:This systematic literature search was conducted using the PubMed, Web of Science, Embase, and Cochrane Library databases. With the inclusion and exclusion criteria, we summarized all of the papers and performed a statistical analyses.Results:In general, the SET-CAN/NUP214 fusion gene is very rare in adult acute leukemia, more frequently found in T-ALL than in other types of leukemia, and more often in males. Flow cytometry data indicated that the markers CD34, CD33, CD13, and CD7 were common in SET-CAN/NUP214 positive acute leukemia, including ALL. Fluorescence in situ hybridization and arrays are important methods for detecting the fusion gene in newly diagnosed patients and can detect chromosomal del(9)(q34) will be detected. The chromosomal karyotype may be normal or complex, and, in terms of survival analysis, transplantation results in a better prognosis than chemotherapy alone.Conclusions and implications of key findings:The presence of SET-CAN/NUP214 fusion gene may be a Minimal Residual Disease of early recurrence, and it might be a poor indicator of outcome.Limitations:The mechanism, clinical characteristics, therapy and prognosis of the SET-CAN/NUP214 fusion gene in hematological malignancies require further research.

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.

Phospho-Specific Flow Cytometry Reveals Signaling Heterogeneity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

Several signaling pathways are aberrantly activated in T-ALL due to genetic alterations of their components and in response to external microenvironmental cues. To functionally characterize elements of the signaling network in T-ALL, here we analyzed ten signaling proteins that are frequently altered in T-ALL -namely Akt, Erk1/2, JNK, Lck, NF-κB p65, p38, STAT3, STAT5, ZAP70, Rb- in Jurkat, CEM and MOLT4 cell lines, using phospho-specific flow cytometry. Phosphorylation statuses of signaling proteins were measured in the basal condition or under modulation with H2O2, PMA, CXCL12 or IL7. Signaling profiles are characterized by a high variability across the analyzed T-ALL cell lines. Hierarchical clustering analysis documents that higher intrinsic phosphorylation of Erk1/2, Lck, ZAP70, and Akt, together with ZAP70 phosphorylation induced by H2O2, identifies Jurkat cells. In contrast, CEM are characterized by higher intrinsic phosphorylation of JNK and Rb and higher responsiveness of Akt to external stimuli. MOLT4 cells are characterized by higher basal STAT3 phosphorylation. These data document that phospho-specific flow cytometry reveals a high variability in intrinsic as well as modulated signaling networks across different T-ALL cell lines. Characterizing signaling network profiles across individual leukemia could provide the basis to identify molecular targets for personalized T-ALL therapy.

Jurkat T cells are immunophenotypically distinct from T-cell acute lymphoblastic leukemia cells due to high-level surface expression of CD5

: Human leukemic T cells show decreased surface CD5 (sCD5) and increased cytoplasmic CD5 (cCD5). When we examined their expressions in the Jurkat T cells, it showed increased sCD5 and decreased cCD5, which is in sharp contrast with the pattern of CD5 expression observed for human leukemic T cells. Furthermore, this opposite pattern was due to the absence of an exonal switch between E1A and E1B. This study suggests that Jurkat cell does not retain all characteristics of T-ALL cells; thus, we should carefully interpret the data obtained using Jurkat T cell as a model cell line of T-ALL.

P341: A NOVEL BISPECIFIC T CELL ENGAGER (UMG2-CD3) IS EFFECTIVE AGAINST CORTICAL-DERIVED ACUTE LYMPHOBLASTIC LEUKEMIA

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived by T cell precursors and characterized by poor prognosis.The immunotherapy has revolutionized the outcome of B cell acute lymphoblastic leukemia (B-ALL), but the absence of tumor-specific T cell antigen hampers its efficacy in T-ALL. Therefore, the development of novel immune-therapeutic options for the management of this orphan disease is eagerly awaited. CD1a is a glycoprotein expressed on cortical T-ALL and only on healthy thymocytes and Langerhans cells. Taking into account its safe pattern of expression, CD1a might represent a valuable therapeutic target for thetreatment of T-ALL. Aims: With the aim to provide an effective immune-therapeutic strategy for T-ALL, we developed a bispecific T cell engager (BTCE) derived from a novel UMG2 mAb that recognize a previously uncharacterized CD1a epitope. Methods: To evaluate the specificity of UMG2 binding to CD1a epitope, HEK293T cells which do not express CD1a endogenously, have been used. In this regards, cells were transfected with a plasmid that encode for CD1a or with an empty vector and UMG2 reactivity has been evaluated by flow cytometry. To assess the unicity of UMG2 mAb binding, a competitive binding assay between UMG2 and commercially available CD1a antibodies has been performed. The UMG2 expression profile on peripheral blood cells from healthy donors and on a panel of cortical T-ALL cells has been evaluated. To develop a UMG2-CD3 construct, an asymmetric 2 + 1 UMG2-CD3 bispecific T cellengager (BTCE) has been generated by using knobs into hole technology. UMG2-CD3 T cell-redirected cytotoxicity has been evaluated on HEK293T wild type, on HEK293T-CD1a+ and on patient-derived T-ALL cells, co-cultured with peripheral blood mononuclear cells (PBMCs), CD4/CD8 depleted and CD56 enriched lymphocytes at different E:T ratio.Moreover, T cell activation has been assesed by flow cytometry. UMG2-CD3 anti-tumor activity against a CD1a+ T-ALL cells has been evaluated in vivo. For this purpose,Hu-PBMCs NSG mouse model has been generated and tumor growth has been assessed by fluorescent imaging probe via IVIS system. Results: UMG2 mAb recognizes a novel CD1a epitope and does not compete with any of the commercially available anti-CD1a mAbs with the exception of a partial competition with NA1/34-HLK clone. A strong UMG2 reactivity has been observed on T-ALL cells, while no binding has been found on normal blood cells. A concentration-dependent T cell cytotoxicity on CD1a+ T-ALL cells co-cultured with PBMCs in the presence of UMG2-CD3 has been observed. Minimal UMG2-CD3 residual anti-tumor activity has been observed in CD4/CD8 depleted and CD56 enriched lymphocytes. CD56 depleted and Fc-blocked BMCs were able to induce an anti-T-ALL activity comparable to total PBMCs, demonstrating that UMG2-CD3 could not recruit monocytes and NK cells through Fc-FcyR interaction. Moreover, the concentration-dependent increase of i) T cell proliferation, ii) cytotoxic degranulation marker (CD107a), iii) expression of cell surface activation markers (CD25, CD69),and iv) pro-inflammatory cytokine secretion (IL-2, TNF-α, IFN-γ) has been observed in the presence of UMG2-CD3. Importantly, in an in vivo immune-humanized NSG mice model,UMG2-CD3 was able to significantly inhibit tumor growth and then conferring the survival advantage of treated animals. Summary/Conclusion: All our findings, demonstrated that UMG2-CD3 BTCE represents a promising immune-therapeutic agent against T-ALL to be further investigated for clinical translation.

P332: MIR-625-5P IS NOVEL CANDIDATE ONCOMIR IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IMPLICATED IN REGULATION OF APOPTOSIS VIA REPRESSION OF HARAKIRI

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL, arising from T-cell precursors. miRNAs are non-coding RNAs, contributing to leukemogenesis by involvement in key cellular functions such as proliferation, apoptosis, and signaling. We previously found in miRNA transcriptome analysis that miR-625-5p is overexpressed in T-ALL patients, thus is a potential oncomiR in T-ALL. Here, we confirm the implication of miR-625-5p in the regulation of apoptosis in T-ALL cells in vitro. We show that it acts via repression of HARAKIRI (HRK). HRK was originally identified as a proapoptotic gene induced by reduced levels of cytokine in hematopoietic cells and repressed by the expression of death-repressor proteins. Although its proapoptotic function is well described, little is known about the post-transcriptional mechanisms that may participate in HRK inactivation. Aims: The aim of this study was to investigate the mechanism of miR-625-5p oncogenic action via unraveling its targetome and functional effects in T-ALL in vitro. Methods: T-ALL JURKAT cells (with high endogenous miR-625-5p level) were transduced for stable inhibition of this miRNA. To evaluate the influence of miR inhibition on growth of T-ALL cells, we used flow cytometry GFP competition assay and CCK8 proliferation assay. To identify the genes mediating the effect of miR-625-5p, we subjected JURKAT cells to Ago2-RIP-seq. We used magnetic beads coated with anti-AGO2 antibody to immunoprecipitate RNA bound to RISC complexes. Ago2-IP and total RNA fractions were sequenced (polyA RNA-seq, 150 bp reads, 60M PE reads/sample, Illumina NovaSeq 6000). Transcripts depleted in Ago2-IP fraction upon miR inhibition were screened for the presence of miR-625-5p binding sites. Luciferase assay was performed to confirm direct interaction between hsa-miR-625-5p and 3’UTR of HRK gene. The changes of HRK protein level upon miR-625-5p inhibition were evaluated via Western Blot. To estimate the apoptosis rate in T-ALL cells we used two flow cytometry assays based on Annexin V and Caspase 3/7 staining. Results: Inhibition of miR-625-5p decreased the growth of JURKAT cells. In RIP-seq analysis we identified 384 transcripts depleted in RISC by at least 20% upon miR-625-5p inhibition and having at least one putative 3’UTR binding site for this miRNA. Overrepresentation analysis performed for these genes indicated their involvement in the apoptotic process. Among transcripts depleted in RIP-seq with predicted binding sites for miR-625-5p, we found HRK gene, previously reported to have proapoptotic activity. We postulate that repression of this gene by overexpressed miR-625-5p contributes to decreased apoptosis and to growth advantage of T-ALL cells. To further confirm that miR-625-5p affects growth of T-ALL cells via negative regulation of apoptosis, we performed apoptotic assays in JURKAT cells and showed that inhibition of this miRNA increased apoptosis rate, in line with our hypothesis on the oncogenic potential of miR-625-5p. We confirmed the direct interaction between miR-625-5p and HRK 3’UTR by luciferase assay. Additionally, in Western blot a subtle (approaching statistical significance) increase of HRK protein upon miR-625-5p inhibition was observed, supporting our notion that this miRNA is indeed a negative regulator of HRK. Image:Summary/Conclusion: miR-625-5p is a novel candidate oncogenic miRNA in T-ALL, implicated in the negative regulation of apoptosis. Post-transcriptional repression of the proapoptotic HRK gene is a putative mechanism contributing to oncogenic properties of this miRNA when overexpressed in T-ALL cells.

P338: BRANCHED CHAIN AMINO ACID TRANSAMINASE 1 ASSOCIATES WITH THE KU70/KU86 HETERODIMER AND MODULATES THE DNA DAMAGE RESPONSE IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Branched Chain Amino Acid Transferase 1 (BCAT1), a cytosolic aminotransferase converts BCAAs into their corresponding branched-chain α-keto acids and vice versa. Aberrant expression of BCAT1 has been demonstrated in different tumor models, including acute leukemia. However, its biological role in T-ALL remains to be elucidated. Aims: The purpose of this study was to dissect the functional role of BCAT1 in T-ALL, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression. Methods: We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (ALL0434 protocol). BCAT1 transcript and protein levels were determined in T-ALL cell lines (n=13) and patient derived xenografts (PDX; n=12). Metabolomics analysis (using 13C6 labeled Leucine) was performed following NOTCH1 inhibition. Further, five PDX samples were treated with a γ secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined by Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Kinetics of the DNA damage response (DDR) was evaluated in BCAT1 knock-down cells. Drug combination experiments using the DNA damaging agent (etoposide) and a specific BCAT inhibitor (ERG245) were performed in vitro and in vivo in PDX T-ALL models. Results: We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Interestingly, NOTCH1 mutated PDX samples also showed higher BCAT1 expression compared to un-mutated cases. Metabolic studies disclosed that BCAAs oxidation is decreased following NOTCH1 inhibition (reduction in 3-methyl-2 oxovaleric acid). Further, inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. ChIP analysis and luciferase reporter assays suggest that NOTCH1 may directly regulate BCAT1 expression. Functionally, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair and rRNA processing. Indeed, we found Ku70/Ku86 proteins to interact with BCAT1. Evaluation of the DDR following DNA damage, disclosed that BCAT1 deficient cells present an accentuated but defective DDR which translates into marked DNA damage leading to pronounced cell death. Consistently, a marked synergistic therapeutic response was found between the DNA damaging drug etoposide and the BCAT specific inhibitor ERG245 in numerous T-ALL models. Summary/Conclusion: NOTCH1 may directly regulate BCAT1 expression in T-ALL cells. BCAT1 is involved in sustaining genomic integrity following DNA damage through its interaction with the Ku70/Ku86 heterodimer, known to mediate classical non-homologous end-joining. Our results identify BCAT1 as a novel therapeutic target and suggest that the combination between DNA double stand break inducing agents (such as etoposide) and a BCAT inhibitor could be particularly useful in NOTCH1-mutant T-ALL cases.

P322: THERAPEUTIC EFFECT OF TARGETING CASEIN KINASE II / WDR5 AXIS IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignance with limit treatment choices and extremely poor prognosis. WD repeat domain 5 (WDR5) is essential for the methyltransferase activity of MLL1 complex and plays an important role in various biological functions. We reported that targeting Casein Kinase II (CK2)/Ikaros axis by CK2 inhibitor CX-4945 has therapeutic efficacy in B-ALL. However, the effect of WDR5 inhibitor and CX-4945 in T-ALL remains unknown. Aims: This study is to explore the synergistic effect of WDR5 inhibitor OICR-9429 and CK2 inhibitor CX-4945 and the potential underlying mechanisms of this novel drug combination in T-ALL. Methods: The expression profile and clinical significance of WDR5 and ATAD2 in T-ALL were explored by analyzing the transcriptome data of T-ALL patients from the GEO database. CCK-8 assay was performed to analyze the synergistic effect of OICR-9429 combined CX-4945 in CEM and MOLT4 cell lines. Cell cycle effect of inhibitors were detected by flow cytometry after 72h cultured. RNA-seq was performed after CEM cells were treated with OICR-9429, CX-4945, and vehicle control for 72 hours. Differential expression genes (DEGs) and pathways were analyzed by R-software. WDR5 and ATAD2 were knockdown by shRNA in CEM and MOLT4 cells. qPCR and Western Bolt were performed to test the DEGs detected by RNA-seq. Results:WDR5 is significantly over-expressed in T-ALL patients compared with healthy controls in two independent cohorts and is associated with shorter overall survival (P<0.01). Bioinformatic analysis revealed that the cell cycle was the most significantly altered pathway between WDR5 high and low expression patients (P<0.001) (Figure 1A). In vitro assay showed that OICR-9429 and CX-4945 alone exhibited an anti-proliferation effect on T-ALL cells after being treated for 72h. In addition, we found that OICR-9429 and CX-4945 had a significant synergistic effect on cell proliferation arrest in CEM (Figure 1B) and MOLT4 cells compared with a single drug (P<0.05). Cell cycle assay revealed that OICR-9429 (Figure 1C) and CX-4945 induced a G1-phase arrest in CEM and MOLT4 cells and this effect was significantly increased upon OICR-9429 and CX-4945 combination (P<0.01) (Figure 1D). The anti-proliferation and cell cycle arrest effects were also exhibited in WDR5 knockdown CEM and MOLT4 cells (P<0.05). Pathway analysis of the DEGs showed that the cell cycle pathway was obviously altered after OICR-9429 and CX-4945 treated (P<0.001) (Figure 1E). In addition, CX-4945 strongly down-regulated WDR5 (Figure 1F) expression in both transcriptome level and protein level, and knockdown of WDR5 increased the anti-leukemic effect of CX-4945 (P<0.01) (Figure 1G), suggesting that WDR5 was the downstream target of CK2 and CX-4945 in T-ALL. Moreover, RNA-seq data showed that ATAD2, an important oncogene was suppressed upon treatment of OICR-9429, CX-4945 (Figure 1F), and WDR5 knockdown (P<0.001). Moreover, the combination of OICR-9429 and CX-4945 strongly induced ATAD2 downregulation compared with either single drug (P<0.001) (Figure 1H). Likewise, we found that ATAD2 is up-regulated in T-ALL patients and associated with poor prognosis and cell cycle pathway abnormal activation (P<0.001). ATAD2 knockdown significantly inhibited cell proliferation and induced a G1-phase cell cycle arrest in T-ALL cells (P<0.01) (Fig 1I). Image:Summary/Conclusion: Our data showed that OICR-9429 and CX-4945 have synergistic efficacy in T-ALL. Dual targeting WDR5 and CK2 may be a potential therapeutic approach for T-ALL through the CK2-WDR5-ATAD2 axis.

STAT5 does not drive steroid resistance in T-cell acute lymphoblastic leukemia despite the activation of BCL2 and BCLXL following glucocorticoid treatment.

Physiological and pathogenic interleukin-7-receptor (IL7R)-induced signaling provokes glucocorticoid resistance in a subset of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Activation of downstream STAT5 has been suggested to cause steroid resistance through upregulation of anti-apoptotic BCL2, one of its downstream target genes. Here we demonstrate that isolated STAT5 signaling in various T-ALL cell models is insufficient to raise cellular steroid resistance despite upregulation of BCL2 and BCL-XL. Upregulation of anti-apoptotic BCL2 and BCLXL in STAT5-activated T-ALL cells requires steroid-induced activation of NR3C1. For the BCLXL locus, this is facilitated by a concerted action of NR3C1 and activated STAT5 molecules at two STAT5 regulatory sites, whereas for the BCL2 locus this is facilitated by binding of NR3C1 at a STAT5 binding motif. In contrast, STAT5 occupancy at glucocorticoid response elements does not affect the expression of NR3C1 target genes. Strong upregulation of BIM, a NR3C1 pro-apoptotic target gene, upon prednisolone treatment can counterbalance NR3C1/STAT5-induced BCL2 and BCL-XL expression downstream of IL7- induced or pathogenic IL7R signaling. This explains why isolated STAT5 activation does not directly impair the steroid response. Our study suggests that STAT5 activation only contributes to steroid resistance in combination with cellular defects or alternative signaling routes that disable the pro-apoptotic and steroid-induced BIM response.

P317: TARGETING HYPERACTIVE PDGFR-B IN T-ALL AND T-LBL

Background: 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. To this end, we have screened a T-ALL/T-LBL patient cohort for copy number variations and identified a novel MYH9-PDGFR-β fusion in a T-LBL case, driving aberrant activation of platelet derived growth factor β (PDGFR-β). Moreover, RNA sequencing data of an independent T-ALL patient cohort showed overexpression of PDGFR-β in the TLX subgroup and in immature T-ALL patients. PDGFR-β is a transmembrane glycoprotein dimer molecule that functions as a receptor tyrosine kinase. Ligand binding results in receptor dimerization and subsequent activation via autophosphorylation, causing downstream activation of various signaling pathways such as PI3K/AKT, MAPK/ERK, JAK/STAT and Notch. 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 and selective PDGFR inhibitor. Mechanistically, CP-673451 inhibits autophosphorylation of dimeric PDGFR-β and in this way prevents phosphorylation of downstream PI3K/AKT and GSK-3α/β. Previous studies showed growth inhibition of multiple tumor xenografts (lung and colon carcinomas) mainly due to a direct antitumor effect. Aims: We aim to assess whether specific PDGFR-β inhibitors can be used as a novel and targeted therapy in T-ALL/T-LBL treatment or if they can be of added value to the current chemotherapy regimen for T-ALL/T-LBL patients. In the future, we would like to investigate the transforming capacity of the MYH9-PDFR-β fusion in an in vitro pro-T cell culture system. Methods: T-ALL cell lines were screened for PDGFR-β levels and cell lines were selected with both high (CTV-1, SEM) and low (MOLT-16, Jurkat, Loucy) PDGFR-β levels. CP-673451 was tested on the selected cell lines. Cell viability was measured using an ATP assay (CellTiter Glo) and downstream signaling pathways were quantified with flow cytometry and western blot. Additionally, spleen cells from PDGFR-βhigh and PDGFR-βlow T-ALL PDX models were treated ex vivo using CP-673451. Results: Both PDGFR-βhigh cell lines and PDGFR-βhigh PDX models showed sensitivity to CP-673451 treatment in nanomolar range, whereas PDGFR-βlow models did not (Fig. 1A-B). Further characterization of CP-673451 treatment showed inhibition of PDGFR-β autophosphorylation both on western blot and flow cytometry. Downstream, phosphorylation levels of STAT3, STAT5 and GSK-3β were decreased upon PDGFR-β inhibition (Fig. 1C). Dephosphorylation of GSK-3β results in activation of this kinase, leading to phosphorylation of MCL1, promoting degradation of this antiapoptotic protein. Interestingly, high MCL1 levels are associated with lowered sensitivity to glucocorticoids, core components of T-ALL/T-LBL treatment. To this end, combination therapy of PDGFR-β inhibition with glucocorticoids can result in synergistic effects. Next, we will conduct in vivo studies to verify if specific PDGFR-β inhibitors could be advantageous in the treatment of PDGFR-βhigh T-ALL/T-LBL cases. Image:Summary/Conclusion: In this study, we have identified MYH9-PDGFRB as a novel rare fusion activating PDGFR-β in a T-LBL patient sample. Moreover, in vitro evaluation in T-ALL cell lines and ex vivo treatment of T-ALL PDX models showed therapeutic benefits from PDGFR-β inhibition by CP-673451.

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.

Abstract 5654: Degradation of Bcl-xL by DT2216 is lethal to T-cell acute lymphoblastic leukemia

Abstract Cancer is a multistep process where genomic and epigenomic alterations results in abnormal cellular proliferation and dissemination. This unregulated cellular proliferation can be interrupted through intervention therapy. One such strategy utilizes proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional small molecular structures consisting of a ligand that binds to a target protein, and upon binding can recruit an E3 ligase which ubiquitinates the target protein, and eventually directs ubiquitinated protein for proteasomal degradation. This has been exploited as a revolutionary therapeutic strategy to degrade oncogenic and/or anti-apoptotic protein(s) that are overly expressed and offer a growth advantage in actively growing tumors. The accumulation of anti-apoptotic proteins creates resistance within the tumor against cancer treatments and preventative strategies in the clinical setting. In the present study, we used DT2216, a PROTAC that targets and promotes degradation of the anti-apoptotic protein Bcl-xL via the VHL (E3 ligase) pathway in T-cell acute lymphoblastic leukemia cells (T-ALL). Unlike other preclinical studies, like ABT263, DT2216 does not cause thrombocytopenia because VHL is poorly expressed in platelets. In our preclinical study, we treated thirteen human T-ALL cells with varying concentrations of DT2216 (0-1 µM) for 24 h and analyzed Bcl-xL expression by western blot analysis. Our data suggests that DT2216 results in the robust degradation of Bcl-xL in a dose-dependent manner at a minimal dose of 10 nM in eight of the cell lines studied (ALL-SIL, P12-Ichikawa, Jurkat, SUP-T1, HBP-ALL, HSB-2, PEER, and DND-41). Bcl-xL degradation triggers apoptosis, which was evident through the presence of cleaved products for Caspase-3 and PARP-1. We examined the expression of other anti-apoptotic proteins, Bcl-2 and Mcl-1 in response to DT2216 and neither protein was changed, which highlights DT2216’s specificity. We further evaluated the effect of DT2216 on the survival of T-ALL cells. Our data suggests that the IC50 for all the T-ALL cell lines ranges from 2-100 nM, with the exception of SUP-T1, which exhibited an IC50 above 1 µM. Our data suggests that DT2216 could serve as a viable, safe, and potent alternative or addition to traditional clinical treatments of T-ALL. Citation Format: Arunima S. Jaiswal, Elizabeth Williamson, Robert A. Hromas, Daohong Zhou. Degradation of Bcl-xL by DT2216 is lethal to T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5654.

Abstract LB015: CCR7-expressing T-cell acute lymphoblastic leukemia mouse model provides a platform for developing novel anti-CCR7 therapies to prevent CNS invasion

Abstract C-C chemokine receptor 7 (CCR7) plays a pivotal role in promoting the migration of T-cell acute lymphoblastic leukemia (T-ALL) cells into the central nervous system during leukemogenesis. Overexpression of CCR7 has also been linked to developing central nervous system (CNS) lymphoma. CCR7 and its ligand, CCL19 are important mediators in the development of these cancers. Thus there is an urgent need for a translational syngeneic CCR7-expressing T-ALL model that can speed up the discovery of drugs that can be used to block CCR7 function during the progression of T-ALL. Using the established ROSA26 floxed-stop Prdm14 (R26PR)/Mx-1 Cre mouse T-ALL model we have initiated studies using a CCR7/CCL19 antagonist, ELC8-83 to examine the molecular mechanisms that mediate CCR7-directed migration to, invasion of, proliferation within and exit from the CNS. mRNA expression of CCR7 in the T-ALL was confirmed by RT-PCR and protein expression on the cell surface was confirmed by flow cytometry. Chemotaxis and calcium mobilization of T-ALL in response to CCL19 was confirmed using transwell-migration assays and FURA-4. ELC8-83 was used to block this signaling. These studies demonstrate the efficacy of ELC8-83 in blocking signaling in T-ALL and provide a platform for testing ELC8-83 in the mouse model of T-ALL. Citation Format: Angel Torres, Aaron Vazquez, Sean Glenn, Zenaida Fuentes, jessica Chacon, Colin Bill, Charlotte M. Vines. CCR7-expressing T-cell acute lymphoblastic leukemia mouse model provides a platform for developing novel anti-CCR7 therapies to prevent CNS invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB015.

Abstract 5337: Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL)

Abstract Background: Inhibitors of apoptosis proteins (IAPs) are overexpressed in ALL leading to resistance to apoptosis and chemo-resistance. Tolinapant, a non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis (cIAP1/2 and XIAP) being evaluated in Phase 1/2 clinical trial [NCT02503423] showed single-agent activity in T-cell lymphoma. Objectives: Herein, we analyzed the preclinical activity of tolinapant in combination with ABT199 and Dexamethasone (DEX) in T-ALL (in vitro and ex-vivo patient derived xenografts (PDX). Results: Using a panel of 8 T-ALL cell lines we analyzed the single agent activity of tolinapant. Loucy and SUP-T11 were most sensitive (IC50 = 190-309 nM). CCRF-CEM and ALL-SIL showed moderate sensitivity (IC50 = 10-18 µM) while Jurkat, MOLT16, MOLT4 and PF382 were resistant. Western blotting (WB) showed decreased levels of cIAP1and cIAP2 with no change in XIAP in response to tolinapant as a single agent. We next investigated the efficacy of tolinapant with Bcl2 inhibitor ABT199. The combination of ABT199 and ASTX660 was synergistic in Loucy cell line with combination index (CI) of 0.14. Cell death was increased to 64±3% in combination v/s 27±0.9% in ABT199. WB showed decreased cIAP2 and increased levels of cleaved caspase 7 and cleaved caspase 9 in combination compared to single agents, suggesting increased apoptosis. Treatment of PDX-derived cells with tolinapant and ABT199 was more effective than monotherapy in inducing apoptosis in CD45+ bulk (46±0.7% to 63±6%, p&amp;lt;0.0001) and leukemia initiating cells (LICs, CD45+ CD7+ CD19- CD34+) (39±3% to 54±8%, p=0.003). Next, we checked the effect of combination of DEX with tolinapant in T-ALL cell lines. CCRF-CEM cell line is from a relapsed patient and is resistant to DEX. The combination was synergistic with CI of 0.26 and cell death of 50±4% compared to 20±3% by DEX. A triple combination of DEX, ABT199 and tolinapant increased apoptotic response to 82.9±1%, compared to DEX+ABT199 dual combination where apoptosis was induced to 52.4±2%. Strong synergy in terms of both cytoreduction as well as apoptosis induction was observed in SUP-T11 cells. Tolinapant sensitized SUP-T11 cells to DEX with ED50 value of 542 nM in combination compared to ~2 µM with DEX treatment. Simultaneous analysis of cell proliferation, stress response and DNA damage using single-cell proteomics analysis showed downregulation of proliferation (Ki-67), stress response (ATF4, LC3B) and increased levels of cleaved PARP, cleaved caspase 3 suggesting increased apoptosis in combination of tolinapant with DEX. Treatment of PDX-derived cells with tolinapant enhanced the cytotoxic effect of DEX in CD45+ bulk (58±2% to 71±0.1%, p&amp;lt;0.0001) and LICs (62±2% to 78±0.9%, p&amp;lt;0.0001). Conclusion: Tolinapant synergizes with the anti-leukemic activity of ABT199 and DEX, establishing a therapeutic rationale for IAP antagonist in T-ALL. Citation Format: Priyanka Sharma, Sujan Piya, Huaxian Ma, Natalia Baran, Muharrem Muftuoglu, Mahesh Basyal, Vivian Ruvolo, George Ward, Tomoko Smyth, Martin J. Sims, Michael Andreeff, Gautam Borthakur. Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5337.