Human T-cell Acute Lymphoblastic Leukemia Cell Lines Research Articles

Therapeutic Effects and Metabolic Rewiring upon Glutaminase Loss in T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid neoplasm that requires intensified chemotherapy for treatment. Despite significant improvements in clinical outcomes with these chemotherapy regimens, still 20-25% of pediatric cases relapse, and the prognosis of these refractory T-ALLs remains extremely poor, underscoring the urgent need to explore novel targets for more effective approaches. The identification of highly prevalent (~60% patients) activating mutations in NOTCH1 has brought considerable interest in developing targeted anti-NOTCH1 therapies, offering promising alternatives to chemotherapy. However, early efforts in the clinic with ϒ-secretase inhibitors (which block a proteolytical cleavage critical for the activation of NOTCH1) have been hampered by limited and heterogenous therapeutic responses. Therefore, further advances in treatment of T-ALL require a deeper understanding of the basic mechanisms driving this disease. We have previously discovered a NOTCH1-controlled metabolic pathway that determines response to anti-NOTCH1 therapies. Specifically, glutaminolysis, which replenishes TCA cycle intermediates to maintain metabolism, is a critical pathway for leukemia cell growth downstream of NOTCH1. As such, inhibition of glutaminolysis pharmacologically or genetically (via deletion of glutaminase, Gls) is highly synergistic with anti-NOTCH1 therapies. However, Gls-deficient T-ALLs eventually progress, highlighting the need for improved therapeutic strategies. Moreover, the glutamine fate and the compensatory mechanisms to overcome Gls loss in leukemic cells in vivo are still poorly understood. To investigate the underlying mechanisms mediating the resistance to Gls loss, we systematically traced the nutrient utilization in isogenic Gls-positive and Gls-negative T-ALLs by intravenously infusing 13C-lactate, 13C-glucose and 13C-glutamine in leukemic mice. Quantitative analysis indicates that Gls-positive T-ALL cells use primarily glutamine as a carbon source to feed the TCA cycle. However, TCA labeling from glutamine decreases in Gls-deficient T-ALL cells, while lactate contribution to label TCA intermediates significantly increases, suggesting a potential compensatory metabolic rewiring mechanism to overcome Gls loss uncoupling TCA from glycolysis. In addition, we observed a reduction of the levels of palmitate, palmitoleate and some polyunsaturated fatty acids, together with a significant downregulation of the expression of Fasn, a central regulator of fatty acid anabolism, in Gls-deficient T-ALL cells. These results suggest a switch to lipid catabolic processes to support leukemic cell growth in absence of Gls in vivo. Gene expression profiling and proteomics analyses of these leukemias with isogenic loss of Gls revealed a strong enrichment in heme/porphyrin-biosynthesis signatures as heme-biosynthetic genes were systematically upregulated in Gls-deficient T-ALL cells as compared to T-ALL control cells. Indeed, using a loss-of function CRISPR-based genetic screening in combination with BPTES, a potent glutaminase inhibitor, in a human T-ALL cell line in vitro, we identified heme-biosynthesis pathway as a potential modulator, whose disruption could sensitize T-ALL cells to glutaminase inhibition. Overall, our findings formally show that while glutaminase plays a key role in T-ALL progression, still cells rapidly rewire their metabolism to compensate for Gls loss demonstrating a marked metabolic flexibility. Moreover, our results have unveiled previously unknown metabolic vulnerabilities that could be exploited in combination with glutaminase inhibitors in the clinical setting with the potential to improve the efficacy of T-ALL therapies and lead to better outcomes for patients.

CCR9 overexpression promotes T-ALL progression by enhancing cholesterol biosynthesis.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy of the lymphoid progenitor cells, contributing to ∼ 20% of the total ALL cases, with a higher prevalence in adults than children. Despite the important role of human T-ALL cell lines in understanding the pathobiology of the disease, a detailed comparison of the tumorigenic potentials of two commonly used T-ALL cell lines, MOLT4 and JURKAT cells, is still lacking. Methodology: In the present study, NOD-Prkdc scid IL2rgd ull (NTG) mice were intravenously injected with MOLT4, JURKAT cells, and PBS as a control. The leukemiac cell homing/infiltration into the bone marrow, blood, liver and spleen was investigated for bioluminescence imaging, flow cytometry, and immunohistochemistry staining. Gene expression profiling of the two cell lines was performed via RNA-seq to identify the differentially expressed genes (DEGs). CCR9 identified as a DEG, was further screened for its role in invasion and metastasis in both cell lines in vitro. Moreover, a JURKAT cell line with overexpressed CCR9 (Jurkat-OeCCR9) was investigated for T-ALL formation in the NTG mice as compared to the GFP control. Jurkat-OeCCR9 cells were then subjected to transcriptome analysis to identify the genes and pathways associated with the upregulation of CCR9 leading to enhanced tumirogenesis. The DEGs of the CCR9-associated upregulation were validated both at mRNA and protein levels. Simvastatin was used to assess the effect of cholesterol biosynthesis inhibition on the aggressiveness of T-ALL cells. Results: Comparison of the leukemogenic potentials of the two T-ALL cell lines showed the relatively higher leukemogenic potential of MOLT4 cells, characterized by their enhanced tissue infiltration in NOD-PrkdcscidIL2rgdull (NTG) mice. Transcriptmoe analysis of the two cell lines revealed numerous DEGs, including CCR9, enriched in vital signaling pathways associated with growth and proliferation. Notably, the upregulation of CCR9 also promoted the tissue infiltration of JURKAT cells in vitro and in NTG mice. Transcriptome analysis revealed that CCR9 overexpression facilitated cholesterol production by upregulating the expression of the transcriptional factor SREBF2, and the downstream genes: MSMO1, MVD, HMGCS1, and HMGCR, which was then corroborated at the protein levels. Notably, simvastatin treatment reduced the migration of the CCR9-overexpressing JURKAT cells, suggesting the importance of cholesterol in T-ALL progression. Conclusions: This study highlights the distinct tumorigenic potentials of two T-ALL cell lines and reveals CCR9-regulated enhanced cholesterol biosynthesis in T-ALL.

Identification of compounds that preferentially suppress the growth of T-cell acute lymphoblastic leukemia-derived cells.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most frequently occurring cancers in children and is associated with a poor prognosis. Here, we performed large-scale screening of natural compound libraries to identify potential drugs against T-ALL. We identified three low-molecular-weight compounds (auxarconjugatin-B, rumbrin, and lavendamycin) that inhibited the proliferation of the T-ALL cell line CCRF-CEM, but not that of the B lymphoma cell line Raji in a low concentration range. Among them, auxarconjugatin-B and rumbrin commonly contained a polyenyl 3-chloropyrrol in their chemical structure, therefore we chose auxarconjugatin-B for further analyses. Auxarconjugatin-B suppressed the in vitro growth of five human T-ALL cell lines and two T-ALL patient-derived cells, but not that of adult T-cell leukemia patient-derived cells. Cultured normal T cells were several-fold resistant to auxarconjugatin-B. Auxarconjugatin-B and its synthetic analogue Ra#37 depolarized the mitochondrial membrane potential of CCRF-CEM cells within 3 h of treatment. These compounds are promising seeds for developing novel anti-T-ALL drugs.

Lyt-200, a Humanized Anti-Galectin-9 Antibody, Exhibits Preclinical Efficacy in Models of Hematological Malignancies

Patients with relapse and refractory (R/R) hematological malignancies typically have 5-year overall survival rates of less than 50% when receiving chimeric antigen receptor (CAR) T-cell therapy and less than 30% after receiving hematopoietic stem cell transplantation (HSCT). Human leukemia cells utilize a variety of biochemical mechanisms, which allow them to escape host immune surveillance. These molecular pathways cause impairment of the anti-cancer activities of immune cells, which could attack and kill leukemia cells. These dismal outcomes for patients with R/R disease highlight the need for novel treatment regimens when current therapeutic options are exhausted. Galectins are s-type lectins that promote diverse biological processes including adhesion, signaling, and immunosuppression. In acute myeloid leukemia (AML), galectin-9 (GAL-9) maintains the leukemia initiating cell (LIC) population and plays an important role in treatment resistance. We recently tested LYT-200, a fully humanized IgG4 αGAL-9 monoclonal antibody, developed by PureTech Health, which has so far been well tolerated with no dose limiting toxicities in a Phase I clinical trial for solid tumors (NCT04666688). We tested LYT-200 in vitro and in vivo across multiple hematological malignancies. Flow cytometry was used to assess the surface expression of GAL-9 on human B-ALL, AML, T-cell acute lymphoblastic leukemia (T-ALL), and diffuse large B-cell lymphoma (DLBCL) cell lines relative to receptor T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) and Programmed cell death protein-1 (PD-1) expression. Compared to healthy human peripheral blood mononuclear cells (PBMCs), where GAL-9 surface expression was low or absent depending on the immune cell, GAL-9 was highly expressed on the surface of all human blood cancer cell lines tested (>100-fold increase). Notably, GAL-9 expression was comparable to PD-1 surface expression and higher than that of TIM-3 on AML, B-ALL, T-ALL, and DLBCL cells. Treating all cell lines with LYT-200 relative to control immunoglobulin resulted in extensive cell death, which occurred after 48 and 72 hours of treatment. Furthermore, a side-by-side comparison with αTIM-3 antibody treatment at equivalent concentrations, revealed that LYT-200 treatment was significantly more effective at killing blood cancer cells at low doses of 10-100 ng/mL in vitro. The in vitro efficacy of LYT-200 against multiple blood cancer subtypes extended to in vivo protection and survival benefit in murine models of T-ALL and AML, both in immunocompromised and immunocompetent mice. When immunocompromised mice were transplanted with human T-ALL cell lines or patient-derived samples, single-agent treatment with LYT-200 (1.5 mg/kg/weekly) was comparable to survival results obtained with methotrexate (0.75 mg/kg/weekly) administration. Notably, the combination of LYT-200 and methotrexate treatments led to the best survival outcomes in both contexts. Similar results were obtained in xenograft studies of AML, where single-agent LYT-200 (1.5 mg/kg/weekly) treatment was more effective at protecting mice than cytarabine (100 mg/kg/weekly). However, combining both treatments resulted in the best survival outcomes with greater than 80% of mice surviving over 2 months after disease appearance. In all, our results demonstrate that GAL-9 is an important driver of multiple blood cancer subtypes and that targeting GAL-9 with LYT-200 represents a potential novel treatment strategy which warrants additional pre-clinical and clinical testing.

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

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

CXCR4 Blockade Targets Progression of Primary T Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL), a malignant neoplasm of immature T cells, is particularly aggressive leukemia with no targeted therapies. Large-scale sequencing efforts to define the genetic make-up of tumors both at diagnosis and during relapse have provided little actionable information for therapeutic development or predict relapse. T-ALL is reliant on the growth factors and chemokines from the bone marrow (BM) microenvironment for its growth, survival, and BM retention. To identify possible vulnerabilities, we have previously examined the role of CXCL12, a chemokine produced by the BM niche, in maintaining NOTCH1 (Notch1-DE) mutation-driven T-ALL 1. Targeting CXCL12/CXCR4 signaling after leukemic onset reduced the T-ALL burden in murine disease models, suggesting that the CXCL12-CXCR4 could be a viable therapeutic target in human T-ALL.To test the therapeutic role of CXCR4 in human T-ALL, we generated CXCR4 knockout T-ALL cell lines using CRISPR-Cas9 editing and characterized their growth and survival in vitro and in vivo. Although the effect of CXCR4 targeting on leukemic growth and apoptosis in vitro was modest (Figure 1A-B), animals engrafted with CXCR4 knockout T-ALL cell lines survived significantly longer than mice injected with control leukemic cells (Figure 1C). To investigate the clinical efficacy of targeting the CXCR4/CXCL12 axis in primary human T-ALL, we generated T-ALL patient-derived xenografts (PDXs) mouse models from 5 T-ALL and 2 Early T-cell precursor acute ALL (ETP-ALL) primary samples. Remarkably, treating PDX mice with MDX1338, a fully humanized anti-CXCR4 antibody2,3, significantly extended survival from all tested PDX models independently of their mutational landscape (Figure 1D-E). This increased survival was accompanied by mobilization of leukemic cells into peripheral blood, suggesting that CXCR4 targeting leads to leukemic disengagement from the BM niche, affecting their survival. Furthermore, BM imaging revealed disengagement of leukemic cells from the BM niche. Collectively, this study provides a pre-clinical rationale for therapeutic interventions targeting CXCR4 in human T-ALL. Figure 1: CXCR4 antagonism suppresses human T-ALL in vivo. A-B) Growth curve (A) and level of Annexin V (B) of human T-ALL cell lines CRISPR/Cas9-edited with sgCXCR4 or control sgOR2W5. C) Kaplan-Meier survival curves of recipient mice transduced with sgOR2W5 or two independent sgCXCR4 are plotted. D) Schematic of treatment with aCXCR4 Ab (MDX-1338) or isotype control. E-F) Kaplan-Meier survival graph (left) and peripheral blood levels of hCD45+ of NSG mice engrafted with primary adult (E) and pediatric (F) T-ALL samples and treated with aCXCR4 Ab or isotype control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD147-specific chimeric antigen receptor T cells effectively inhibit T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is invasive and heterogeneous, and existing therapies are sometimes unsuccessful. Chimeric antigen receptor (CAR) T cell therapy is a breakthrough tumor treatment method, particularly for B cell acute lymphoblastic leukemia. We found that CD147 was highly expressed in tumor T cells of T-ALL patients and T cell lymphoma. Therefore, CD147-CAR T cells that contain a humanized single-chain variable fragment targeting human CD147 and a second-generation CAR frame were constructed for treating T-ALL. CD147-CAR T cells were able to maintain a healthy proliferation rate, preserving a subset of CD62L+/CCR7+ memory T cells. CD147-CAR T cells showed a potent anti-tumor activity against human T-ALL cell line and T-ALL blasts, releasing high level of cytokines in the process. However, CD147-CAR T cells exhibited potential safety toward human normal cells and CD147-deficent cells. NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice were used to establish a T-ALL xenograft model and CD147-CAR T cells conferred robust protection against T-ALL progression and significantly improved survival in mice. Overall, we found that CD147 is a potential antigen target of CAR T cell therapy for T-ALL.

T-ALL can evolve to oncogene independence.

The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor.

Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) drives migration and progression of T-cell acute lymphoblastic leukemia in vitro and in vivo

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer. There are no immunotherapies and few molecularly targeted therapeutics available for treatment of this malignancy. The identification and characterization of genes and pathways that drive T-ALL progression are critical for the development of new therapies for T-ALL. Here, we determined that the protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) plays a critical role in T-ALL initiation and progression by promoting leukemia cell migration. PRL-3 is highly expressed in patient T-ALL samples at both the mRNA and protein levels compared to normal lymphocytes. Knock-down of PRL-3 expression using short-hairpin RNA (shRNA) in human T-ALL cell lines significantly impeded T-ALL cell migration capacity in vitro and reduced their ability to engraft and proliferate in vivo in xenograft mouse models. Additionally, PRL-3 overexpression in a Myc-induced zebrafish T-ALL model significantly accelerated disease onset and shortened the time needed for cells to enter blood circulation. Reverse-phase protein array (RPPA) and gene set enrichment analysis (GSEA) revealed that the SRC signaling pathway is affected by PRL-3. Immunoblot analyses validated that manipulation of PRL-3 expression in T-ALL cells affected the SRC signaling pathway, which is directly involved in cell migration, although Src was not a direct substrate of PRL-3. More importantly, T-ALL cell growth and migration were inhibited by small molecule inhibition of PRL-3, suggesting that PRL-3 has potential as a therapeutic target in T-ALL. Taken together, our study identifies PRL-3 as an oncogenic driver in T-ALL both in vitro and in vivo and provides a strong rationale for targeted therapies that interfere with PRL-3 function.

Protein Translocation Inhibitors Overcome Cytokine-Induced Glucocorticoid Resistance in T-Cell Acute Lymphoblastic Leukemia

Glucocorticoids (GCs) are central to the treatment of T-cell acute lymphoblastic leukemia (T-ALL), and upfront resistance to GCs is a poor prognostic factor. We previously demonstrated that over one-third of primary patient T-ALLs are resistant to the GC dexamethasone (DEX) when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the microenvironment of leukemic blasts and that plays a well-established role in leukemogenesis. Mechanistically, we demonstrated that GCs paradoxically induce their own resistance by promoting the upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augments signal transduction through the JAK/STAT5 axis, ultimately leading to increased STAT5 transcriptional output. This promotes the upregulation of the pro-survival protein BCL-2, which opposes DEX-induced apoptosis. Given that IL7-induced GC resistance depends on de novo synthesis of IL7R in response to DEX, and that newly synthesized IL7R reaches the cell surface via trafficking through the secretory pathway, we hypothesized that inhibiting the translocation of nascent IL7R peptide into the secretory pathway would effectively overcome IL7-induced DEX resistance. Sec61 is a protein-conducting channel in the membrane of the endoplasmic reticulum (ER) that is required for the cotranslational insertion of nascent polypeptides into the ER upon recognition of the signal sequence on secreted and cell surface proteins. To test the hypothesis that Sec61 inhibition could overcome IL7-induced DEX resistance, we utilized the human T-ALL cell line CCRF-CEM, which recapitulates the resistance phenotype observed in primary patient samples. Using a series of structurally distinct small molecule inhibitors of the Sec61 translocon, we demonstrated that Sec61 inhibition effectively overcomes the increase in cell surface IL7R expression in response to DEX. This occurs despite a persistent elevation in IL7R transcript expression following DEX exposure, confirming that Sec61 inhibitors act post-transcriptionally to attenuate cell surface IL7R expression. To determine whether the sensitivity of IL7R to Sec61 inhibitors is due specifically to the interaction between the IL7R signal sequence and Sec61 inhibitors, we generated IL7R constructs containing hydrophobic amino acid substitutions in the signal sequence, which are predicted to confer resistance to Sec61 inhibitors. Upon transient transfection of these constructs into HEK293T cells, we found that these mutations rendered IL7R resistant to the effects of Sec61 inhibition, confirming that the IL7R signal sequence confers sensitivity to these inhibitors. Using the Bliss independence model of synergy in CCRF-CEM cells, we demonstrated that Sec61 inhibitors potently synergize with DEX to overcome IL7-induced DEX resistance. Importantly, at concentrations at which synergy occurs, Sec61 inhibitors demonstrate no single-agent effect on cell survival, suggesting that these effects are not due to an overall reduction in secretory and membrane protein biogenesis. Furthermore, Sec61 inhibitors failed to sensitize CCRF-CEM cells to other chemotherapies used in T-ALL, none of which demonstrate IL7-induced resistance, thereby suggesting that these effects on DEX sensitivity are due specifically to the reduction in cell surface IL7R. To determine if Sec61 inhibitors prevent the DEX-induced increase in STAT5 transcription, we analyzed BCL-2 expression in cells exposed to DEX and IL7, and found that Sec61 inhibitors attenuate the increase in BCL-2 expression in a dose-dependent manner. We next analyzed a cohort of 34 primary patient T-ALL samples. As in CCRF-CEM cells, we found that specifically in those samples with IL7-induced DEX resistance, Sec61 inhibitors synergized with DEX to induce cell death in the presence of IL7. This effect occurred concomitantly with a reduction in cell surface IL7R expression and BCL-2 expression. Taken together, these data demonstrate the efficacy and feasibility of Sec61 inhibition as a novel and rational therapeutic strategy to overcome the IL7-induced DEX resistance phenotype that affects over one-third of newly diagnosed T-ALL patients. Disclosures Sharp: Kezar Life Sciences: Patents & Royalties. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Taunton:Global Blood Therapeutics: Equity Ownership; Principia Biopharma: Equity Ownership, Patents & Royalties; Cedilla Therapeutics: Consultancy, Equity Ownership; Pfizer: Research Funding; Kezar Life Sciences: Equity Ownership, Patents & Royalties, Research Funding.

PRE‐T CELL RECEPTOR SIGNALING DRIVES LEUKEMOGENESIS AND IS A THERAPEUTIC TARGET IN T CELL ACUTE LYMPHOBLASTIC LEUKEMIA

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid neoplasm that results from the malignant transformation of T-cell progenitors. The biology of T-ALL is strongly influenced by its developmental origins. For example, mutations in NOTCH1, a critical regulator of thymocyte development, are found in approximately 60% of patient tumors. Less is known, however, about the extent to which other determinants of thymocyte development participate in the leukemogenesis of T-ALL. Using a thymus transplantation-based, spontaneous mouse model of T-ALL, we found that multiple β-selection checkpoint factors were upregulated in leukemic T cells, including Ptcra, a subunit of the pre-T cell receptor (pre-TCR) complex. Genetic ablation of Ptcra in the mouse model dramatically reduced the occurrence of T-ALL. In human T-ALL cell lines, CRISPR/Cas9 knockout of PTCRA reduced in vitro proliferative capacity and the ability to form tumors in vivo. Analysis of clinical T-ALL datasets and patient samples demonstrated that PTCRA is highly and specifically expressed in leukemic T cells but not in normal, mature T cells, suggesting an appropriate therapeutic window for targeted therapy in T-ALL. Cumulatively, our findings highlight an important role for pre-TCR signaling in driving and sustaining T-ALL and support the further evaluation of PTCRA-directed therapies in T-ALL. Keywords: Notch pathway; T-cells. Disclosures: Kuhnert, F: Employment Leadership Position: Regeneron Pharmaceuticals Inc.

PS918 CIRCRNA DEREGULATION IN B-CELL PEDIATRIC ACUTE LEUKEMIA: THE MLL RECOMBINOME AND BEYOND

Background: Activating NOTCH1 mutations are present in approximately 60% of T-cell acute lymphoblastic leukemia (T-ALL) cases. Mutant NOTCH1 is strictly dependent on γ-secretase processing for activation thereby providing a strong rationale for the use of γ-secretase inhibitors (GSIs) as a therapeutic approach for T-ALL. However, available GSI's lead to severe “on-target” gastrointestinal tract, skin and thymus toxicity, limiting their therapeutic application and clinical translation. What is less appreciated is that the enzymatic activity of the γ-secretase complex reflects the combined activity of at least four different complexes. These complexes broadly differ by the inclusion of one of two Presenilin (PSEN) proteins: PSEN1 or PSEN2, providing an opportunity to target specific complexes whilst avoiding toxicity in otherwise healthy tissue. Aims: To determine whether subunit selective gamma secretase inhibition is an effective therapeutic strategy in T-cell acute lymphoblastic leukemia. Methods: RNA-seq analysis was undertaken on T-ALL cell lines and patient samples. To generate mice carrying a Psen1 deletion in all committed B and T cell progenitors, we crossed Psen1 conditional knockout mice with CD2Cre. To generate conditional inducible Psen1 knockout mice, we bred animals harboring the conditional Psen1 allele with Rosa26Cre-ERt2 mice. In vivo bone marrow transplant (BMT) assays were performed using isolated hematopoietic stem and progenitor (HSPCs) cells and transduced with mutant NOTCH1 (HD-mutant, delta PEST) retroviral vectors. T-ALL patient derived xenograft studies in NSG mice were treated daily with MRK-560 (30 μmol/kg) and leukemia burden assessed by bioluminescence and human CD45 staining. Gut toxicity was assessed by Periodic acid–Schiff (PAS) immunohistochemistry. Results: Here we show that PSEN1 is expressed significantly higher than PSEN2 in human T-ALL cell lines and primary T-ALL samples. Genetically, the conditional deletion of Psen1 in mice significantly decreased mutant NOTCH1 receptor processing but did not impair normal T-cell development. Furthermore, specific inducible deletion of Psen1 in a mutant NOTCH1-driven leukemia significantly reduced leukemia burden and increased overall survival in vivo. The significant decrease in leukemic burden identified through genetic loss of Psen1 then prompted us to investigate whether selective pharmacological PSEN1 inhibition would be a viable strategy for T-ALL treatment. To this end, we tested the PSEN1 selective inhibitor MRK-560, which has ∼100-fold selectivity over PSEN2, on four different T-ALL patient-derived xenograft samples with varying NOTCH pathway mutations. MRK-560 monotherapy treatment significantly decreased leukemia burden as measured by in vivo bioluminescence and human CD45 in the peripheral blood and significantly increased overall survival. Moreover, treatment with MRK-560 did not cause any pathological changes in the gastrointestinal architecture nor increased numbers of secretory goblet cells, assessed by Periodic Acid-Schiff (PAS) staining, even after 4 weeks of treatment. Summary/Conclusion: These results demonstrate that inhibition of Presenilin-1 is highly effective in decreasing leukemia whilst avoiding dose-limiting toxicities and that Presenilin-1 selective compounds provide a new therapeutic strategy for safe and effective targeting of T-ALL.

Beta1 integrin blockade overcomes doxorubicin resistance in human T-cell acute lymphoblastic leukemia

Growing evidence indicates that cell adhesion to extracellular matrix (ECM) plays an important role in cancer chemoresistance. Leukemic T cells express several adhesion receptors of the β1 integrin subfamily with which they interact with ECM. However, the role of β1 integrins in chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL) is still ill defined. In this study, we demonstrate that interactions of human T-ALL cell lines and primary blasts with three-dimensional matrices including Matrigel and collagen type I gel promote their resistance to doxorubicin via β1 integrin. The blockade of β1 integrin with a specific neutralizing antibody sensitized xenografted CEM leukemic cells to doxorubicin, diminished the leukemic burden in the bone marrow and resulted in the extension of animal survival. Mechanistically, Matrigel/β1 integrin interaction enhanced T-ALL chemoresistance by promoting doxorubicin efflux through the activation of the ABCC1 drug transporter. Finally, our findings showed that Matrigel/β1 interaction enhanced doxorubicin efflux and chemoresistance by activating the FAK-related proline-rich tyrosine kinase 2 (PYK2) as both PYK2 inhibitor and siRNA diminished the effect of Matrigel. Collectively, these results support the role of β1 integrin in T-ALL chemoresistance and suggest that the β1 integrin pathway can constitute a therapeutic target to avoid chemoresistance and relapsed-disease in human T-ALL.

The actin‐binding protein cortactin controls transendothelial migration and organ infiltration of acute lymphoblastic leukemia T‐cells

T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive neoplasm due to high rates of organ infiltration and relapse, which are major obstacles for curing the disease. Infiltration of T‐ALL cells requires extravasation, a migratory process highly dependent on actin cytoskeleton dynamics. Cortactin is an actin‐binding protein that regulates actin remodeling and thus cell migration. High expression of cortactin is associated with invasiveness in several types of solid tumors, and recently, expression of cortactin in adult B‐cell chronic lymphocytic leukemia was correlated with negative prognostic factors. However, it is unknown whether cortactin plays a role in T‐ALL biology. Here, we analyzed cortactin expression in the murine and human T‐ALL cell lines 6645/4 and Jurkat, and cortactin functions using different in vitro and in vivo methods such as transendothelial migration, bone marrow (BM) colonization and xenotransplantation assays. Compared to normal T cells, leukemic T cells expressed significantly higher levels not only of cortactin but also of the integrin VLA‐4 and CXCR4, the receptor for the T cell chemokine CXCL12, which are both required for T cell extravasation. Leukemic T cells transmigrated significantly more, and cortactin levels were significantly higher in cells capable of transmigrating across endothelial monolayers towards CXCL12. Of note, only leukemic T cells with highest cortactin levels were capable of invading BM‐like organoids. Preliminary in vivo assays showed that leukemic T cells had different levels of cortactin depending on the infiltrated organ. Surprisingly, once leukemic T cells completed infiltration they diminished cortactin expression suggesting that cortactin is needed for initial organ invasion, but not for subsequent functions. Taken together, our results highlight an important role of cortactin in T‐ALL infiltration; and contribute to a better understanding of T‐ALL aggressiveness. Thus, cortactin may be a potential pharmacological target to prevent T‐ALL organ infiltration.Support or Funding InformationCONACyTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Co-operation of ABT-199 and gemcitabine in impeding DNA damage repair and inducing cell apoptosis for synergistic therapy of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia with limited therapeutic options available. Here, we evaluated the therapeutic potential of the combination of the Bcl-2 antagonist ABT-199 and cytotoxic agent gemcitabine in T-ALL cell lines. Our results showed that the combination of ABT-199 and gemcitabine exhibited synergistic cytotoxicity and induced significant apoptosis in human T-ALL cell lines (Jurkat and Molt4). The augmented apoptosis induced by combination treatment was accompanied by the greater extent of mitochondrial depolarization and enhanced DNA damage. Importantly, single agent induced DNA damage alone but did not inhibit RAD51/BRCA1-mediated repair for DNA double-strand breaks. In contrast, the combination of ABT-199 and gemcitabine disrupted RAD51/BRCA1-dependent DNA repair and remarkably activated caspase-3 and PARP to trigger apoptosis. Moreover, ABT-199 exerted an antagonistic action towards Bcl-2 and Bcl-xL, but to a certain extent moderately increased Mcl-1 level that could be compromised by gemcitabine. In conclusion, our study showed that the combination of ABT-199 and gemcitabine exhibited synergistic cytotoxicity in T-ALL cells by cooperatively targeting DNA damage repair pathway and Bcl-2 family proteins.

NOTCH1 Represses MCL-1 Levels in GSI-resistant T-ALL, Making them Susceptible to ABT-263.

Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL. We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both in vitro experiments and in vivo studies on patient-derived xenograft models. We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax in vitro and leads to tumor regressions in vivo. Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.

Glucocorticoids Paradoxically Induce Intrinsic Steroid Resistance through a STAT5-Mediated Survival Mechanism in T-Cell Acute Lymphoblastic Leukemia

▪Upfront resistance to glucocorticoids (GCs) confers a poor prognosis for children with T-cell acute lymphoblastic leukemia (T-ALL). Using primary diagnostic samples from the Children's Oncology Group trial AALL1231, we previously demonstrated that one-third of patient T-ALL samples are intrinsically resistant to GCs when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the T-ALL microenvironment. Furthermore, we demonstrated that inhibiting JAK/STAT signaling downstream of the IL7 receptor (IL7R) with the JAK1/2 inhibitor ruxolitinib (RUX) overcomes GC resistance in these samples. The objective of the present study was to determine the mechanism of IL7-induced GC resistance in T-ALL and to identify novel therapeutic targets to enhance GC sensitivity. We utilized CCRF-CEM cells, a human T-ALL cell line, as a model system in conjunction with primary patient samples. Exposing CCRF-CEM cells to IL7 induced phosphorylation of STAT5, the predominant downstream effector of IL7R signaling. When cultured in the presence of IL7 and the GC dexamethasone (DEX), CCRF-CEM cells recapitulated the IL7-induced GC resistance phenotype observed in patient samples. This resistance could be overcome with RUX, and Bliss index analysis demonstrated a synergistic relationship between DEX and RUX in the presence of IL7. Furthermore, CRISPR/Cas9 mediated knockout of STAT5 (STAT5 KO) was sufficient to overcome resistance, implicating STAT5 as the critical mediator of IL7-induced GC resistance. DEX exposure potently induced upregulation of IL7R expression in CCRF-CEM cells. Using a luciferase reporter construct containing a series of STAT5 response elements, we demonstrated that in the presence of IL7, DEX-induced upregulation of IL7R expression is associated with increased downstream signal transduction, leading to a significant increase in STAT5 transcriptional output. We then performed RNA-seq to further assess the functional consequences of this enhanced STAT5-mediated transcription. Gene set enrichment analysis (GSEA) revealed that STAT5 target genes were significantly upregulated in cells exposed to DEX and IL7 relative to IL7 alone (normalized enrichment score -2.27; p < 0.001; FDR < 0.001), suggesting that DEX exposure augments activation of the STAT5 transcriptional program. One critical component of this program that was induced by the combination of DEX and IL7 was the anti-apoptotic family member BCL2, which was not induced by DEX alone. Further analysis of its protein expression in CCRF-CEM cells confirmed this paradoxical upregulation of BCL2 specifically by the combination of DEX and IL7. Furthermore, BCL2 was not upregulated by DEX and IL7 in STAT5 KO cells, consistent with this being a STAT5-mediated effect. IL7-induced GC resistance could be overcome with shRNA-mediated knockdown of BCL2 and with pharmacologic inhibition of BCL2 by venetoclax. Similar to the effect observed with RUX, Bliss index analysis demonstrated synergy between DEX and venetoclax in the presence of IL7. Consistent with our observations in CCRF-CEM cells, an analysis of primary diagnostic T-ALL samples revealed DEX-induced upregulation of IL7R expression in samples with IL7-induced GC resistance, which was associated with increased BCL2 expression in the presence of DEX and IL7. Finally, we performed a similar analysis in healthy murine thymocytes, and found that CD4/CD8 double negative (DN) and CD4 or CD8 single positive (SP) thymocytes, but not double positive (DP) thymocytes, exhibited profound IL7-induced GC resistance that was associated with DEX-induced upregulation of IL7R expression and increased BCL2 expression in the presence of DEX and IL7. These data are consistent with the known role of IL7 specifically at the DN and SP stages of development, and suggests that IL7-induced GC resistance is a physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALL samples. Taken together, these data demonstrate that GCs paradoxically induce their own resistance in a subset of T-ALLs and in normal developing T-cells by augmenting a STAT5-mediated pro-survival program that results in upregulation of BCL2. Furthermore, we demonstrate that inhibition of JAK/STAT signaling or of BCL2 may have considerable therapeutic benefit to enhance GC sensitivity in T-ALL patients with IL7-induced GC resistance. DisclosuresTeachey:La Roche: Consultancy; Amgen: Consultancy.

Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease.The in vitro component of this synthetic system affords direct access to “pre-leukemia” cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the “HOXB high” subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and “unknown” transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models.The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly “evolved” samples. DisclosuresSteidl:Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

The CXCR4 Antagonist, BL8040, Is Highly Active Against Human T-ALL in Preclinical Models

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. Approximately 20-25% of pediatric and 50% of adult patients with T-ALL relapse following induction therapy, and the prognosis after relapse is dismal, with 3-year event-free survival of only 10-15%. Thus, there is a clear unmet clinical need for better therapies for T-ALL. CXCL12 (stromal-derived factor-1, SDF1) is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. Here we explore the hypothesis that inhibition of the CXCR4 signaling with a potent new CXCR4 antagonist, BL-8040 alone, or in combination with ABT263 (navitoclax), a BCL2/BCLXL antagonist, will have therapeutic activity against T-ALL.To test this hypothesis, we xenotransplanted a human T-ALL cell line (P12/Ichikawa cells) or 5 different patient-derived T-ALL xenografts into non-irradiated NSG mice. In each case, the T-ALL cells were allowed to engraft and then mice were randomly assigned to one of four treatment groups: 1) vehicle control; 2) BL-8040 alone; 3) ABT263 alone; 4) combined BL-8040 and ABT263. Treatment was given for 2 weeks, and leukemia burden monitored weekly by bioluminescent imaging or by flow cytometry to quantify human CD45+ T-ALL cells in the blood. A minimum of 10 mice in each cohort from two independent experiments were analyzed (Figure 1). A significant reduction in T-ALL burden was observed in all T ALL samples after treatment with BL8040 alone, with a mean fold-decrease compared with control mice after 2 weeks of treatment of 966 ± 651 (range 16-3,486). Consistent with a prior report, the response to ABT263 alone was more variable, with responses seen in 4 of 6 T-ALL samples. All T-ALL samples had a marked response to the combination of BL-8040 and ABT263, with a mean fold decrease of 4,389 ± 2,602 (range, 139-15,199). A previous study suggested that inhibition of CXCR4 signaling may suppress Myc expression (Pitt et al, Cancer Cell, 2015), which is relevant, since overexpression of Myc secondary to mutations in the Notch pathway are common in T-ALL. However, we observed no difference in Myc protein expression or expression of Myc target genes following BL-8040 treatment. Consistent with this observation, treatment with BL8040 had no impact on the cell cycle status of T-ALL cells in vivo. Of note, we observed a marked decrease in Akt and Erk phosphorylation following BL8040, suggesting that CXCR4 signaling may regulate T ALL survival in vivo by suppressing these pathways.Conclusion. Collectively, these data suggest that BL-8040, either alone or in combination with ABT263, is highly active in T-ALL and support our ongoing clinical trial of BL-8040 in combination with nelarabine for patients with relapsed T-ALL (NCT02763384). [Display omitted] DisclosuresUy:GlycoMimetics: Consultancy; Curis: Consultancy. Vainstein:Biolinerx: Employment. Sorani:BioLineRx Ltd.,: Employment. Bohana-Kashtan:BioLineRx Ltd.,: Employment, Equity Ownership; Cell Cure Neurosciences: Equity Ownership. Shaw:BioLineRx Ltd.: Employment, Equity Ownership.

USP7 deubiquitinates and stabilizes NOTCH1 in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive leukemia that is primarily caused by aberrant activation of the NOTCH1 signaling pathway. Recent studies have revealed that posttranslational modifications, such as ubiquitination, regulate NOTCH1 stability, activity, and localization. However, the specific deubiquitinase that affects NOTCH1 protein stability remains unestablished. Here, we report that ubiquitin-specific protease 7 (USP7) can stabilize NOTCH1. USP7 deubiquitinated NOTCH1 in vivo and in vitro, whereas knockdown of USP7 increased the ubiquitination of NOTCH1. USP7 interacted with NOTCH1 protein in T-ALL cells, and the MATH and UBL domains of USP7 were responsible for this interaction. Depletion of USP7 significantly suppressed the proliferation of T-ALL cells in vitro and in vivo, accompanied by downregulation of the NOTCH1 protein level. Similarly, pharmacologic inhibition of USP7 led to apoptosis of T-ALL cells. More importantly, we found that USP7 was significantly upregulated in human T-ALL cell lines and patient samples, and a USP7 inhibitor exhibited cell cytotoxicity toward primary T-ALL cells, indicating the clinical relevance of these findings. Overall, our results demonstrate that USP7 is a novel deubiquitinase that stabilizes NOTCH1. Therefore, USP7 may be a promising therapeutic target in the currently incurable T-ALL.