T-cell Acute Lymphoblastic Leukemia Cell Research Articles

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

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

Metabolic Profiling as an Approach to Differentiate T-Cell Acute Lymphoblastic Leukemia Cell Lines Belonging to the Same Genetic Subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor mainly affecting children and adolescents. It is driven by multiple genetic mutations that together define the leukemic phenotype. Interestingly, based on genetic alterations and/or deregulated expression, at least six genetic subgroups have been recognized. The TAL/LMO subgroup is one of the most represented genetic subgroups, characterizing 30-45% of pediatric T-ALL cases. The study of lipid and metabolic profiles is increasingly recognized as a valuable tool for comprehending the development and progression of tumors. In this study, metabolic and lipidomic analysis via LC/MS have been carried out on four T-ALL cell lines belonging to the TAL/LMO subgroup (Jurkat, Molt-4, Molt-16, and CCRF-CEM) to identify new potential metabolic biomarkers and to provide a subclassification of T-ALL cell lines belonging to the same subgroup. A total of 343 metabolites were annotated, including 126 polar metabolites and 217 lipid molecules. The statistical analysis, for both metabolic and lipid profiles, shows significant differences and similarities among the four cell lines. The Molt-4 cell line is the most distant cell line and CCRF-CEM shows a high activity in specific pathways when compared to the other cell lines, while Molt-16 and Jurkat show a similar metabolic profile. Additionally, this study highlighted the pathways that differ in each cell line and the possible enzymes involved using bioinformatic tools, capable of predicting the pathways involved by studying the differences in the metabolic profiles. This experiment offers an approach to differentiate T-ALL cell lines and could open the way to verify and confirm the obtained results directly in patients.

Abstract 4752: Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling

Abstract Introduction Relapsed acute lymphoblastic leukemia (ALL) portends a poor prognosis. Glucocorticoids (GCs) are a central component of a multi-agent regimen to treat ALL. GCs mediate ligand-dependent global transcriptional changes activating feedback loop that increases GC receptor (GR) expression and pro-apoptotic signaling. GC resistance is a common mechanism in relapsed/refractory ALL. Altered function of Enhancer of Zeste Homolog 2 (EZH2), a histone methyl transferase (HMT), including increased expression and activating mutations have been reported in ALL. Previously, EZH2 inhibitors have been shown to increase GC efficacy in non-ALL B-cell lymphoid malignancies including non-Hodgkin lymphoma. Furthermore, EZH2 inhibitors reversed GC resistance and increased efficacy of GCs in an ALL subtype with an activating point mutation in an HMT called nuclear set domain 2. Overcoming GC resistance by novel therapeutic strategies may help improve outcomes in relapsed/refractory ALL. Hypothesis EZH2 inhibitors may enhance GC efficacy by augmenting downstream effects of GCs. Methods We performed in-vitro studies of EZH2 inhibitors, EPZ-6438 and CPI-1205, at graded doses in GC resistance human-derived B-cell (SUPB15) and T-cell (SUPT1, KOPTK1, PEER) ALL cell lines followed by graded doses of dexamethasone (dex). Cell viability was performed using CellTiter-Glo assay. Induction of apoptosis was measured with Annexin V flow cytometry. Pro-apoptotic BIM mRNA and protein levels were measured using real-time PCR and immunoblot. Global H3K27me3 protein levels were assessed using immunoblot. Results GC resistant B-cell ALL and T-cell ALL treated with EZH2 inhibitors for 3-7 days followed by the addition of dex for 48-72 hours led to decreased cell viability and increased apoptosis of the ALL cells compared to cells treated with vehicle (DMSO) and dex alone. EZH2 inhibitors on their own did not cause apoptosis. SynergyFinder revealed Bliss synergy scores between 23- 31 in B-cell ALL and T-cell ALL cell lines with greater than 10 considered a synergistic drug interaction. Immunoblotting of cells treated with EZH2 inhibitors alone showed an increase in pro-apoptotic protein, BIM. BIM level was further enhanced when dex was added in combination with EZH2 inhibitors compared to vehicle and dex alone suggesting that EZH2 inhibitors were augmenting BIM expression. Further assessment of the mechanism of how EZH2 inhibitors lead to improved GC efficacy will be performed by integrating transcriptomic and epigenomic profiling in the presence and absence of EZH2 inhibitors. Furthermore, in-vivo testing of EZH2 inhibitors in patient derived xenografts will be performed. Conclusion EZH2 inhibitors can overcome GC resistance in ALL posing as a potential novel therapeutic agent to treat relapsed/refractory ALL. Citation Format: Mansi Dalal, Joanna Yi, Jianping Li, Amin Shobh, Daphne DupereRicher, Richard Bennett, Jonathan Licht. Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4752.

Abstract 5920: Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy

Abstract BACKGROUND: Relapse of T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL) post-chemotherapy or post-immunotherapy remains a therapeutic problem warranting novel therapeutic approaches. Aberrant Wnt pathway activation is a common feature in ALL as Wnt activation through β-catenin is involved in self- renewal of drug resistant leukemia cells. A therapeutical approach to target Wnt signaling in T-ALL and B-ALL remains elusive. Here, we focus on the effect of transducin β-like protein 1 (TBL1) inhibitor tegavivint in combination with chemotherapy in T-ALL and B-ALL. We hypothesize that interrupting the binding of TBL1 to β-catenin abrogates leukemia cell survival and test if tegavivint sensitizes these cells to chemotherapeutic agents such as VDL (Vincristine, Dexamethasone, L-asparaginase) as part of preclinical evaluation of tegavivint in T- and B-cell ALL. METHODS: Four T-ALL cell lines one patient-derived T-ALL case, B-ALL cell lines and eight patient-derived B-ALL cases were used in vitro. We performed cytotoxicity studies using Annexin V/7AAD stainings and flow cytometry or Trypanblue counts to exclude dead cells, western blots, and co-immunoprecipitation assays to investigate effects of TBL-1 inhibition using tegavivint on β-catenin-mediated signaling. Tegavivint was injected intraperitoneally along with chemotherapeutic drugs. RESULTS: β-catenin and TBL1 were uniformly expressed in all T-ALL and B-ALL cells as determined by western blotting, albeit at different levels. Tegavivint decreases β-catenin protein expression as seen through western blot as well as decreases the binding of TBL1 to β-catenin as seen through co-immunoprecipitation (co-IP) in both the T-ALL and B-ALL samples. We also determined that tegavivint as a monotherapy induced apoptosis in both cell lines and patient samples at as little of a dose of 5nM after 24 hours of incubation using Annexin-V/DAPI staining by flow cytometry. We observed that tegavivint can sensitize all cases to chemotherapy (V: Vincristine, D: Dexamethasone, L:L-Asparaginase or Peg-Asparaginase) within 24 hours of incubation. Lastly, in vivo evaluation of tegavivint showed that tegavivint alone and in combination with VD chemotherapy significantly extends survival of NSG mice engrafted with patient-derived T-ALL and B-ALL compared to control treated mice. CONCLUSION: Our preliminary data shows that T-ALL and B-ALL are sensitive to TBL1-β-catenin inhibition using tegavivint as a monotherapy. Tegavivint can also sensitize T-ALL and B-ALL to chemotherapy (VDL). As our data supports our hypothesis that TBL-1-β-catenin inhibition is a new target in ALL therapy, further studies are in progress to preclinically evaluate this approach for clinical care. Citation Format: Samantha Hurwitz, Rachel Friedmann, Tatiana Fourfouris, Ki Jun Lee, Zesheng Wan, Chintan Parekh, Fariba Navid, Deepa Bhojwani, Kimberly Holloway, Yong-Mi Kim. Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5920.

Abstract 3057: The TARZN complex binds to de novo enhancer mutations and promotes oncogenic expression in T-ALL

Abstract TAL1 is overexpressed in 40-60% of T-cell acute lymphoblastic leukaemia (T-ALL) cases and forms an oncogenic core regulatory circuit (CRC) with other transcription factors such as LMO1, LMO2 and GATA3. In 5% of T-ALL cases an insertion of a consensus GT dinucleotide is observed upstream of the TAL1 gene, termed the “Mutation of TAL1 enhancer” (MuTE), driving TAL1 overexpression. Using an in vitro reconstitution DNA pull-down assay combined with quantitative mass spectrometry, we identified nine candidate proteins that preferentially bound to the MuTE sequence compared to the WT sequence. Among the candidates, we demonstrate by reciprocal immunoprecipitation experiments coupled to quantitative mass spectrometry that the RNA methyltransferase TARBP1 and the zinc finger proteins ZBTB2, ZBTB25 and ZNF639 formed a complex that we term TARZN. Interestingly, the TARZN complex also bound to de novo super enhancer sites upstream of the LMO1 and LMO2 genes in T-ALL cells, indicating a putative common mechanism between these different non-coding driver mutations. Furthermore, knock-down of all TARZN members resulted in lower TAL1 protein expression in MuTE-positive but not in MuTE-negative T-ALL cells. Overall, these data suggest that the TARZN complex cooperatively promotes oncogenic expression in T-ALL. Citation Format: Nurkaiyisah Zaal Anuar, Chai Yeen Goh, Boon Haow Chua, Shi Hao Tan, Joana R. Costa, Marc R. Mansour, Takaomi Sanda, Dennis Kappei. The TARZN complex binds to de novo enhancer mutations and promotes oncogenic expression in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3057.

Abstract 2823: Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological malignancy, is characterized by abnormal thymocyte proliferation, frequently with mutations of the Notch-1 gene and has an overall 10-year survival rate of ~27%. A marker of poor prognosis is the presence of T-ALL within the central nervous system (CNS), which is treated with intrathecal chemotherapy and cranial irradiation; two toxic treatments with high morbidities. Gene-expression profiling of Notch-1-induced adhesion regulators showed that C-C chemokine receptor 7 (CCR7) was highly expressed and was associated with leukemic T-cell invasion of the CNS. Normal functions of CCR7 include lymph node homing and immune cell trafficking in response to CCR7 ligands, CC-chemokine ligand 19 (CCL19) or CCL21. In several studies CCL19 has been linked to T-cell invasion of the CNS across the blood-brain barrier (BBB). We hypothesize that antagonizing CCR7 binding to CCL19 will block migration of T-ALL into the CNS and thereby alleviate the need for toxic therapies. To test our hypothesis, we are using receptor internalization assays, calcium mobilization assays, transwell migration assays and an in vitro model of an intact human BBB. We have used flow cytometry-based CCR7 internalization assays ± CCR7 antagonist and found that we can completely block CCL19-induced receptor internalization in human CCR7-expressing CCRF-CEM, and HuT-78 T-ALL cells, and chemotaxis via β1 integrins across a fibronectin-coated transwell membrane. Our BBB employs HBEC-5i, human cerebral microvascular endothelial cells, in transwell chambers coated with gelatin and fibronectin for the adhesion of endothelial cells. HBEC-5i cells are a suitable cell line to make a 3D model of the BBB, as they form an endothelial monolayer with tight junctions (TJ), which is crucial to maintaining the integrity and inhibiting permeability of the BBB. We are establishing a transwell migration assay to assess the migration of CCRF-CEM T-ALL cells across the HBEC-5i cell monolayer. The integrity of the HBEC-5i cell monolayer is assessed by trans-epithelial electrical resistance (TEER), which measures the electrical resistance across the HBEC-5i monolayer, a measure of TJ integrity. Using TEER measurements, we observe that HBEC-5i form a monolayer that reaches an intact monolayer functioning with TJs over 72-96 hours. We are using this model system to investigate the migration of CCR7-expressing CCRF-CEM cells through the BBB ± CCL19. Subsequently, we will add the CCL19 antagonist (CCL197-77) to determine if inhibiting CCR7 is sufficient to eliminate migration of the CCRF-CEM cells through the BBB. If successful, this model will allow us to test the contribution of CCR7 signaling to integrins, the contribution of other immune cell types and the overall molecular mechanisms that mediate invasion of the CNS in a 3-D model of the BBB. Citation Format: Alondra Rodriguez, Cesar I. Cardona, Colin A. Bill, Charlotte M. Vines. Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2823.

Abstract 1733: SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia

Abstract The SET-NUP214 (S/N) fusion gene, which results from either cryptic t(9:9) (q34;q34) or del(9) (q34.11q34.13), is found in a very narrow spectrum of patients diagnosed with Acute Myeloid Leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL). AML patients with S/N fusion were reported to have relatively poor prognostic outcomes despite receiving myeloablative conditioning. While previously, the fusion has been described to overrepresent HOX cluster genes, the underlying mechanism of gene regulation that contributes to the pathology remains elusive due to the rare incidence of the disease. The current study used the MEGAL (ACC719, DSMZ) cell line, identified as the solo megakaryocytic AML line expressing the S/N-fusion, for determining genome-wide DNA methylation (DNAm) and gene expression using 935K Infinium MethylationEPIC v2.0 BeadChip array and RNA-sequencing, respectively. The differential DNAm and expression data were analyzed and compared to the hematopoietic stem progenitor cells (HSPC) or normal megakaryocytes (MK). The ChIP-sequencing data from the S/N positive T-ALL cell line LOUCY has been used to represent the coincidence of regulatory chromatin marks and differentially methylated CpG (mC) sites. We observed n=1934 genes upregulated and n=2396 downregulated in MEGAL, compared to both HSPC and MK. The direction of expression changes between MEGAL and HSPC and MEGAL and MK showed a high correlation (r=0.92, p< 0.01). Per the previous literature, we observed an overrepresentation of HOX genes (n=19) in MEGAL. Based on DNAm profiling, we noticed a propensity of hypermethylation in MEGAL across the promoters (93%±0.6), bodies (93%±0.8), and intergenic (85%±0.3) regions, compared to HSPC/MK. Next, we integrated the mC and expression at the promoters and identified that the majority (75%±3) of the genes belong to the hyper-down (n=2563 against HPSC; n=1907 against MK) or hyper-up (n=965 against HPSC; n=923 against MK) clusters. We further investigated the HOXB (HOXB7, HOXB8, HOXB9) and HOXC (HOXC10, HOXC12, HOXC13, HOXC13-AS, HOXC4, HOXC5, HOC6, HOXC8, HOXC9, HOXC-AS1, HOXC-AS2, and HOXC-AS3) cluster genes, where hypermethylated CpGs mainly were concentrated at the CpG islands and overlapped with active promoter (H3K4me3, H3K4me1) signatures or enhancer-like (CTCF bound) elements. We also observed frequent overlaps between mC and H3K36me3 across the observed genes. The differentially expressed genes in this AML subtype were enriched for the hippo-signaling pathway, calcium signaling, focal adhesion, and PI3-Akt pathways. Collectively, our data showed that S/N favors genome-wide hypermethylation, which, in conjunction with the cis-regulatory elements, induces overrepresentation of the HOX cluster genes. These novel epigenetic vulnerabilities are worth pursuing for investigating the cellular function and pathophysiology of the disease. Citation Format: Samrat Roy Choudhury, Akhilesh Kaushal. SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1733.

Abstract 5845: Cytarabine resistance leads to a unique mutational signature

Abstract The prognosis of recurrent T-cell acute lymphoblastic leukemia (T-ALL) is poor, mainly due to chemotherapy resistance. The mechanism(s) leading to chemoresistance at relapse are incompletely understood. We previously characterized a mouse strain that is hypomorphic for the DNA replication factor Mcm2. Mcm2hypo mice develop T-ALL due to acquired DNA copy number variations (CNV) involving tumor suppressor or oncogenes. We hypothesize that treatment of Mcm2hypo cells with specific chemotherapy agents may reveal CNV that lead to chemoresistance. We treated two Mcm2hypo T-ALL cell lines (designated 2883 and 2869) and an Mcm2 WT T-ALL cell line (designated 7298) with cytarabine (ARAC), an active agent often incorporated in T-ALL treatment regimens. After one year of treatment with gradually increasing ARAC concentrations, we obtained ARAC resistant cell lines (designated with the suffix CR) that tolerate drug concentrations up to 10,000X that of parental cell lines. These samples were characterized by sparse WGS (for CNV assessment), RNA-Seq and WES. Both the 2883CR and 2869CR cell lines had bi-allelic mutations involving Dck, which is the rate-limiting enzyme in the cytidine salvage pathway, and is thought to be required for metabolism of ARAC. In both cases, one allele was deleted while the second allele had a splice site/region mutation leading to partial intron retention and resultant frameshift. The 7298CR cell line had homozygous deletion of Dck. Western blotting showed absence of WT Dck protein in all three cell lines. Further analysis of WES data revealed thousands of acquired single nucleotide variants (SNV) in both Dck mutant Mcm2hypo cell lines. These mutations occurred in a specific trinucleotide context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, and C>G in a GCC context) generating a mutational signature that is not present in the COSMIC database. In addition, the parental 2883 and 2869 cell lines showed fewer mutations (ie, hundreds vs thousands) in a similar context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, but not the C>G in GCC context). A GEMINI (Genotoxic Mutational Signature Identified After Clonal Expansion In Vitro) assay of untreated 2883CR and 2869CR cells demonstrated that ongoing ARAC exposure was not required for the mutational signature. We hypothesize that the basic signature (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context) can be produced by Mcm2 deficiency, and this signature is amplified and modified (to include a C>G in GCC context) by Dck inactivation. In summary, we identified three mechanisms responsible for Dck inactivation associated with ARAC resistance: copy number loss, splice site mutations, and splice region mutations. Moreover, Dck inactivation leads to a specific mutation signature. These findings add to emerging data that exposure to specific chemotherapy agents can lead to specific mutational signatures. Citation Format: Dengchao Cao, Mianmian Yin, Zhenhua Zhang, Taimour Baslan, Jack Zhu, Ryan Bertoli, Paul Meltzer, Peter Aplan. Cytarabine resistance leads to a unique mutational signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5845.

In vivo activity of the second-generation proteasome inhibitor ixazomib against pediatric T-cell acute lymphoblastic leukemia xenografts

The overall survival rate of patients with T-cell acute lymphoblastic leukemia (T-ALL) is now 90%, although patients with relapsed T-ALL face poor prognosis. The ubiquitin-proteasome system maintains normal protein homeostasis, and aberrations in this pathway are associated with T-ALL. Here we demonstrate the in vitro and in vivo activity of ixazomib, a second-generation orally available, reversible, and selective proteasome inhibitor against pediatric T-ALL cell lines and patient-derived xenografts (PDXs) grown orthotopically in immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJAusb (NSG) mice. Ixazomib was highly potent in vitro, with half-maximal inhibitory concentration (IC50) values in the low nanomolar range. As a monotherapy, ixazomib significantly extended mouse event-free survival of five out of eight T-ALL PDXs in vivo.

The BET inhibitor GNE-987 effectively induces anti-cancer effects in T-cell acute lymphoblastic leukemia by targeting enhancer regulated genes.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy originating from T progenitor cells. It accounts for 15% of childhood and 25% of adult ALL cases. GNE-987 is a novel chimeric molecule developed using proteolysis-targeting chimeras (PROTAC) technology for targeted therapy. It consists of a potent inhibitor of the bromodomain and extraterminal (BET) protein, as well as the E3 ubiquitin ligase Von Hippel-Lindau (VHL), which enables the effective induction of proteasomal degradation of BRD4. Although GNE-987 has shown persistent inhibition of cell proliferation and apoptosis, its specific anti-tumor activity in T-ALL remains unclear. In this study, we aimed to investigate the molecular mechanisms underlying the anti-tumor effect of GNE-987 in T-ALL. To achieve this, we employed technologies including RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and CUT&Tag. The degradation of BET proteins, specifically BRD4, by GNE-987 has a profound impact on T-ALL cell. In in vivo experiments, sh-BRD4 lentivirus reduced T-ALL cell proliferation and invasion, extending the survival time of mice. The RNA-seq and CUT&Tag analyses provided further insights into the mechanism of action of GNE-987 in T-ALL. These analyses revealed that GNE-987 possesses the ability to suppress the expression of various genes associated with superenhancers (SEs), including lymphoblastic leukemia 1 (LCK). By targeting these SE-associated genes, GNE-987 effectively inhibits the progression of T-ALL. Importantly, SE-related oncogenes like LCK were identified as critical targets of GNE-987. Based on these findings, GNE-987 holds promise as a potential novel candidate drug for the treatment of T-ALL.

Thymic-Epithelial-Cell-Dependent Microenvironment Influences Proliferation and Apoptosis of Leukemic Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological cancer characterized by the infiltration of immature T-cells in the bone marrow. Aberrant NOTCH signaling in T-ALL is mainly triggered by activating mutations of NOTCH1 and overexpression of NOTCH3, and rarely is it linked to NOTCH3-activating mutations. Besides the known critical role of NOTCH, the nature of intrathymic microenvironment-dependent mechanisms able to render immature thymocytes, presumably pre-leukemic cells, capable of escaping thymus retention and infiltrating the bone marrow is still unclear. An important challenge is understanding how leukemic cells shape their tumor microenvironment to increase their ability to infiltrate and survive within. Our previous data indicated that hyperactive NOTCH3 affects the CXCL12/CXCR4 system and may interfere with T-cell/stroma interactions within the thymus. This study aims to identify the biological effects of the reciprocal interactions between human leukemic cell lines and thymic epithelial cell (TEC)-derived soluble factors in modulating NOTCH signaling and survival programs of T-ALL cells and TECs. The overarching hypothesis is that this crosstalk can influence the progressive stages of T-cell development driving T-cell leukemia. Thus, we investigated the effect of extracellular space conditioned by T-ALL cell lines (Jurkat, TALL1, and Loucy) and TECs and studied their reciprocal regulation of cell cycle and survival. In support, we also detected metabolic changes as potential drivers of leukemic cell survival. Our studies could shed light on T-cell/stroma crosstalk to human leukemic cells and propose our culture system to test pharmacological treatment for T-ALL.

BACH2-mediated CD28 and CD40LG axes contribute to pathogenesis and progression of T-cell lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL characterized by its high heterogeneity and unfavorable clinical features. Despite improved insights in genetic and epigenetic landscapes of T-ALL, the molecular mechanisms that drive malignant T-cell development remain unclear. BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription repressor recognized as a tumor suppressor in B-cell malignancies, but little is known about its function and regulatory network in T-ALL. Here we found extremely low levels of BACH2 in T-ALL clinical samples and cell lines compared to normal T cells. Overexpression of BACH2 in T-ALL cells not only induced cell growth retardation but also inhibited cancer progression and infiltration in xenografts. Further RNA sequencing (RNA-seq) analysis revealed significant alterations in regulation of defense and immune responses in T-ALL cells upon BACH2 overexpression. Strikingly, CD28 and CD40LG, two essential stimulatory molecules on T cells, were for the first time identified as novel downstream targets repressed by BACH2 in T-ALL cells. Interestingly, both CD28 and CD40LG were indispensable for T-ALL survival, since largely or completely silencing CD28 and CD40LG led to rapid cell death, whereas partial knockdown of them resulted in cell-cycle arrest and enhanced apoptosis. More importantly, BACH2-mediated CD28 and CD40LG signals contributed to cell migration and dissemination of T-ALL cells to the bone marrow, thus adding a new layer to the BACH2-mediated tumor immunoregulation in T-cell malignancies.

CAD204520 Targets NOTCH1 PEST Domain Mutations in Lymphoproliferative Disorders.

NOTCH1 PEST domain mutations are often seen in hematopoietic malignancies, including T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), splenic marginal zone lymphoma (SMZL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL). These mutations play a key role in the development and progression of lymphoproliferative tumors by increasing the Notch signaling and, consequently, promoting cell proliferation, survival, migration, and suppressing apoptosis. There is currently no specific treatment available for cancers caused by NOTCH1 PEST domain mutations. However, several NOTCH1 inhibitors are in development. Among these, inhibition of the Sarco-endoplasmic Ca2+-ATPase (SERCA) showed a greater effect in NOTCH1-mutated tumors compared to the wild-type ones. One example is CAD204520, a benzimidazole derivative active in T-ALL cells harboring NOTCH1 mutations. In this study, we preclinically assessed the effect of CAD204520 in CLL and MCL models and showed that NOTCH1 PEST domain mutations sensitize cells to the anti-leukemic activity mediated by CAD204520. Additionally, we tested the potential of CAD204520 in combination with the current first-line treatment of CLL, venetoclax, and ibrutinib. CAD204520 enhanced the synergistic effect of this treatment regimen only in samples harboring the NOTCH1 PEST domain mutations, thus supporting a role for Notch inhibition in these tumors. In summary, our work provides strong support for the development of CAD204520 as a novel therapeutic approach also in chronic lymphoproliferative disorders carrying NOTCH1 PEST domain mutations, emerging as a promising molecule for combination treatment in this aggressive subset of patients.

Zebrafish drug screening identifies Erlotinib as an inhibitor of Wnt/β-catenin signaling and self-renewal in T-cell acute lymphoblastic leukemia

The Wnt/β-catenin pathway’s significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential. However, the clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex cross-talk of Wnt signaling with other pathways. In this study, we leveraged a zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/β-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/β-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which rely on active β-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafish’s translational potential in drug discovery and repurposing and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/β-catenin signaling.

Development of PD-1 blockade peptide-cell conjugates to enhance cellular therapies for T-cell acute lymphoblastic leukemia.

Blockade of the interaction of the immune checkpoint receptor programmed cell death protein (PD)-1 and its ligand PD-L1 has been found to be a promising cancer treatment. Our previous studies identified that nABPD1 competed with PD-L1 to bind PD-1. The aim of this study was to evaluate the efficacy and safety of anti-tumor immunotherapy of ICIK cells conjugated with peptides in vivo and in vitro. Here, we synthesized the nABPD1 derivatives SBP1 and SBP2 and showed that their binding efficiency to PD-1-positive improving cytokine-induced killer (ICIK) cells was 98 and 82%, respectively. The cytotoxicity of ICIK cells to T-cell acute lymphoblastic leukemia (T-ALL) cells was increased by conjugating with SBP1 or SBP2, which was 2 times higher than that of ICIK cells alone. Furthermore, mice experiments showed that the fluorescence intensity of leukemia cells in T-ALL xenograft models was reduced by more than 95%, indicating that the peptides enhanced the therapeutic effect in vivo, while morphological evaluations showed that the peptides had no toxicity to important organs. Therefore, peptide-cell conjugates (PCCs) may be a novel method to improve the efficacy of cancer immunotherapy by blocking PD-1 in T-ALL patients.

CASZ1 upregulates PI3K-AKT-mTOR signaling and promotes T-cell acute lymphoblastic leukemia.

CASZ1 is a conserved transcription factor involved in neural development, blood vessel assembly and heart morphogenesis. CASZ1 has been implicated in cancer, either suppressing or promoting tumor development depending on the tissue. However, the impact of CASZ1 on hematological tumors remains unknown. Here, we show that the T-cell oncogenic transcription factor TAL1 is a direct positive regulator of CASZ1, that T-cell acute lymphoblastic leukemia (T-ALL) samples at diagnosis overexpress CASZ1b isoform, and that CASZ1b expression in patient samples correlates with PI3KAKT- mTOR signaling pathway activation. In agreement, overexpression of CASZ1b in both Ba/F3 and T-ALL cells leads to the activation of PI3K signaling pathway, which is required for CASZ1b-mediated transformation of Ba/F3 cells in vitro and malignant expansion in vivo. We further demonstrate that CASZ1b cooperates with activated NOTCH1 to promote T-ALL development in zebrafish, and that CASZ1b protects human T-ALL cells from serum deprivation and treatment with chemotherapeutic drugs. Taken together, our studies indicate that CASZ1b is a TAL1-regulated gene that promotes T-ALL development and resistance to chemotherapy.

Ubiquitin-Mediated Degradation of MORF4L1 By FBXO11 Suppresses Cholesterol Biosynthesis in T Cell Acute Lymphoblastic Leukemia

The substrate receptor of SKP1-Cullin1-FBOX complex, FBXO11, is frequently mutated in both diffuse large B cell lymphoma and acute myeloid leukemia, and typically functions as a tumor suppressor by targeting substrates for ubiquitin-mediated degradation. Although FBXO11 is also mutated in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines, the underlying mechanism in T-ALL development remains to be established. Here, we identified that both endogenous activation and exogenous overexpression of FBXO11 could suppress the growth of T-ALL cell in vitro and in vivo. While conversely, loss of FBXO11 accelerates the T-ALL cell proliferation. Transcriptomic and total quantitative proteomic analyses in FBXO11-depleted T-ALL cells reveal activation of the cholesterol biosynthesis pathway with accumulation of intracellular cholesterol levels. Importantly, we identified a chromatin regulator MORF4L1, mortality factor 4 like protein 1, as a functional substrate of FBXO11 in T-ALL cells. We first observed that FBXO11 exhibited frequent copy number alterations in T-ALL cell lines and in patient cohorts from public datasets. Moreover, by CRISPR SAM (Synergistic Activation Mediator) endogenous activation and lentiviral mediated gene overexpression, we validated that FBXO11 played a role in inhibiting tumor cell proliferation in T-ALL cells both in vitro and in vivo. Conversely, loss of FBXO11 accelerated T-ALL cell proliferation. Taken together, our results suggest that FBXO11 functions as a tumor suppressor in T-ALL. Mechanistically, we uncovered that the absence of FBXO11 could activate the cholesterol synthesis signaling pathway from both the RNA-seq and the TMT proteomic analysis in FBXO11-depleted MOLT4 cells compared to control cells. Concordantly, loss of FBO11 resulted in the accumulation of intracellular cholesterol level, suggesting that FBXO11 may suppress cholesterol biosynthesis in T-ALL cells. To understand how FBXO11 controls cholesterol biosynthesis, we further identified the potential substrates from the combined quantitative transcriptome and proteome analysis. MORF4L1, a chromatin modifier involved in histone acetylation, was identified as a key substrate of FBXO11 based on its upregulation at the protein level in FBXO11-depleted cells. Moreover, our data suggest MORF4L1 is a regulator of cholesterol biosynthesis in T-ALL. The inhibition of MORF4L1 suppressed the cellular growth of T-ALL cell lines, and led to the downregulation of the expression of cholesterol-related genes, such as gene HMGCR and HMGCS1. Of note, suppression of MORF4L1 and its cholesterol biosynthesis target genes such as HMGCR, HMGCS1 could rescue the accelerated growth effect after loss of FBXO11, indicating a functional role for MORF4L1 in T-ALL development. In summary, our data support a model wherein the degradation of MORF4L1 by FBXO11 suppresses leukemia cell growth by controlling cholesterol biosynthesis. The identification of this regulatory mechanism is required for T-ALL and provides valuable insights into the metabolic vulnerabilities of leukemia.

CXCR3/CXCL10 Signaling Pathway Drives T-ALL into the CNS/Meningeal Microenvironment

Cure rates for T-cell acute lymphoblastic leukemia (T-ALL) have improved significantly, but central nervous system (CNS) involvement leads to a poor prognosis. Understanding the mechanisms underlying CNS involvement could lead to more effective therapies. The CXCR3/CXCL10 signaling pathway mediates T cell movement across the blood-CSF barrier during neuroinflammation, but its role in T-ALL colonization of the CNS/meninges remains unclear. In our study, we hypothesized that T-ALL cells exploit normal T-cell function to infiltrate the meninges. We examined CXCR3 expression in thymic CD4+CD8+ double positive (DP) cells of ∆E-NOTCH1 T-ALL and control mice. Levels of CXCR3 were higher in CD4 +CD8 + DP cells of T-ALL mice compared to controls. Furthermore, leukemic cells in the meninges, bone marrow (BM), and thymus had higher CXCR3 levels compared to other organs ( p < 0.01), suggesting an organ-specific CXCR3 expression in infiltrating T-ALL cells. Additionally, primary T-ALL samples (n = 24) had elevated CXCR3 mRNA levels compared to normal thymocytes. Using CRISPR-Cas9, we deleted CXCR3 in the KOPTK1 and PER117 cell lines and in two primary patient samples. CXCR3 deletion resulted in decreased proliferation, delayed cell cycle progression in S/G2/M phases but did not affect apoptotic cell death. CXCR3 inactivation led to decreased activation of AKT, ERK1/2, JNK, and reduced the expression of cell motility regulators. Notably, T-ALL cells migrated more efficiently to CXCL10 than to CXCL9 and CXCL11, which was reduced upon CXCR3 silencing ( p < 0.01). Mechanistically, we demonstrated that USP7, a ubiquitin-specific protease, regulates and stabilizes the CXCR3 protein via deubiquitination. To explore the role of CXCR3 in T-ALL in vivo, we transplanted CXCR3 KO and control KOPTK1 cells into NSG mice (8 mice/group). Mice with CXCR3 KO had increased survival and significantly lower leukemia burden in the BM and extramedullary tissues compared to the control group ( p < 0.01). We next analyzed the levels of CXCR3 ligands, CXCL9, CXCL10, and CXCL11, in the blood serum and CSF of ∆E-NOTCH1 and control mice (5 mice/group). T-ALL mice had elevated CXCL10 levels in both blood serum and CSF, with higher levels in the CSF, suggesting compartmentalization of the immune response between the CSF and blood ( p < 0.01). CXCL9 and CXCL11 expression were minimal or undetectable. Remarkably, upon injecting ∆E-NOTCH1-transformed hematopoietic progenitors into CXCL10 knockout ( CXCL10 KO) and control B6 mice (5 mice/group), tissue analysis revealed a significant decrease in T-ALL meningeal infiltration in the CXCL10 KO mice, but not in other organs ( p < 0.01). Conversely, control mice exhibited high levels of leukemic cells in all tested organs. We found that meningeal stromal cells (PDGFR+, CD13+, CD31-, CD45-) expressed CXCL10 in ∆E-NOTCH1 mice, unlike hematopoietic (CD45+) and endothelial cells (CD31+, CD45-), compared to control mice. We further observed upregulation of CXCL10 in human meningeal stromal cells co-cultured with KOPTK1, PER117, and primary samples. Treatment with a CXCR3 antagonist or knockout of CXCR3 reduced leukemic cell migration to the meningeal stromal cells compared to controls ( p < 0.01). Conversely, migration of T-ALL cells to the tested stromal cells was reduced upon treatment with a CXCL10 neutralizing antibody or by CXCL10 deletion in stromal cells. Additionally, we identified that the CXCR3/CXCL10 signaling pathway regulated the expression of VLA-4 and VCAM-1, promoting T-ALL cell adhesion to the meningeal stroma. Genetic inactivation or pharmacological inhibition of CXCR3 in T-ALL cells or CXCL10 in meningeal stromal cells reduced cell-cell adhesion and the expression of VLA-4 and VCAM-1 in T-ALL and meningeal stroma, respectively, further supporting the role of CXCL10-CXCR3 in regulating T-ALL adhesion. Lastly, we investigated whether inflammatory cytokines derived from T-ALL induce CXCL10 in the meningeal microenvironment. We found elevated expression of Ifng, Tnf and Il27 in T-ALL cells infiltrating the meninges, but not other organs of ΔE-NOTCH1 mice ( p < 0.01). Silencing these genes in human T-ALL cells resulted in reduced CXCL10 expression and secretion, along with reduced leukemic cell migration to the tested meningeal stromal cells. In summary, our study emphasizes the key role of CXCR3-CXCL10 signaling in T-ALL progression and meningeal infiltration.

Suppression of Super-Enhancer-Driven TAL1 Expression By KLF4 in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by differentiation arrest and clonal proliferation of immature thymocytes. Many oncogenes in T-ALL are genes encoding transcription factors. TAL bHLH transcription factor 1 ( TAL1) is one of the most frequently dysregulated transcription factors in T-ALL and is ectopically overexpressed in approximately 50% of T-ALL cases, owing to chromosome translocations, SIL (SCL-interrupting locus) -TAL1 fusion, and 5‘ super-enhancer (SE)-generating mutations. TAL1 overexpression caused by 5‘ TAL1 SE is found in 5% of patients with T-ALL and is associated with unfavorable clinical outcomes. However, no clinically available drugs specifically inhibit TAL1 or TAL1 SE components. Kruppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors that exhibits both oncogenic and tumor-suppressive functions in a context-dependent manner. In T-cell malignancies, KLF4 is regarded as a tumor suppressor because KLF4 expression is silenced by promoter methylation, and the induction of KLF4 promotes apoptosis through the BCL-2/BCL-XL pathway. However, whether KLF4 affects the expression of T-ALL-related transcription factors remains unclear. We first analyzed three independent previously reported microarray datasets to explore the essential downstream targets of KLF4 in T-ALL cells. The following groups of genes were extracted: genes upregulated in patients with T-ALL compared to normal thymocytes (GSE46170), genes upregulated in Klf4-deficient T-ALL mice (GSE75663), and genes downregulated in KLF4-overexpressing human T-ALL cells (Li W et al. Mol. Cancer. 2015). Overlapping genes in these groups were identified as potential downstream targets of KLF4. Among these, we focused on TAL1, a well-known oncogenic transcription factor gene in T-ALL. We performed a functional analysis of KLF4 using TAL1-positive T-ALL cell lines (Jurkat, MOLT-3, CCRF-CEM, and RPMI-8402). Genetic induction of KLF4 showed stronger anti-leukemic effects in Jurkat and MOLT-3 cells, 5‘ TAL1 SE-positive T-ALL cell lines, compared with CCRF-CEM and RPMI-8402 cells, SIL-TAL1-positive T-ALL cell lines. KLF4 overexpression significantly reduced mRNA expression levels of TAL1 in Jurkat and MOLT-3 cells but not in CCRF-CEM and RPMI-8402 cells. Thus, we hypothesized that KLF4 specifically suppressed SE-driven TAL1 expression by impairing mutated TAL1 enhancer activity. The luciferase reporter assay confirmed that the activity of the mutated TAL1 enhancer was significantly reduced by the exogenous expression of KLF4, whereas that of the wild-type reporter was not affected. According to previous studies, mutations in TAL1 enhancers in Jurkat and MOLT-3 cells create MYB primary motifs, and MYB binding at this location is essential for the formation of 5‘ TAL1 SE. Therefore, we examined whether KLF4 represses MYB expression. The chromatin immunoprecipitation assay and qPCR analysis revealed that KLF4 bound to the proximal gene promoter of MYB and directly downregulated its expression. The luciferase reporter assay using the MYB promoter showed a significant reduction of reporter activity upon KLF4 overexpression. Finally, we investigated whether pharmacological induction of KLF4 using APTO-253, a small-molecule inducer ofKLF4, could control leukemia. APTO-253 inhibited the growth of all TAL1-positive T-ALL cell lines at nanomolar concentrations. The GI 50 values of APTO-253 for Jurkat and MOLT-3 cells were lower than those for CCRF-CEM and RPMI-8402 cells, which was consistent with the KLF4-overexpression-induced growth inhibition of T-ALL cell lines. APTO-253 induced KLF4 expression and decreased TAL1 and MYB expression in Jurkat and MOLT-3 cells. Furthermore, exogenous MYB expression rescued growth inhibition by APTO-253. Collectively, these results indicated that APTO-253 suppressed the growth of T-ALL cells by targeting SE-driven TAL1 expression via the induction of KLF4. In summary, we revealed that KLF4 suppressed SE-driven TAL1 expression via the direct inhibition of MYB. Moreover, pharmacological induction of KLF4 demonstrated anti-leukemic activity in T-ALL cells. These findings propose a promising strategy for patients with T-ALL and 5' TAL1 SE.

The Transcription Factors NFATc1 and NFATc2 Control Glucocorticoid Resistance in Pediatric T-Cell Acute Lymphoblastic Leukemia

Background: The overall survival of pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) is significantly improved (˜75-80%) with the current therapeutic approaches. Nevertheless, for those patients that do not respond to conventional treatment and experience relapse, the prognosis is extremely poor. In this context, it is well defined that resistance to glucocorticoids (GCs), pillar drugs in the treatment protocol, can predispose pediatric T-ALL patients to a poor outcome. A pivotal role of LCK kinase has been elucidated in supporting GC resistance in T-ALL cells in our previous study and recently confirmed by other colleagues. However, to date, the biological processes modulated by LCK in this context are not yet defined. Aim: the identification of new mechanisms underlying GC resistance can lead to alternative therapeutic approaches to prevent or overcome GC resistance and ameliorate the outcome of this subgroup of patients. Methods and Results: we uncovered the involvement of NFATc1 and NFATc2 transcription factors, that act downstream LCK kinase, in guiding GC resistance in T-ALL cells. Of note, a high NFATc1 and NFATc2 transcriptional activity characterizes pediatric GC resistant T-ALL patients at diagnosis, that in turns show a low Glucocorticoid Receptor (GR) activity. In agreement, NFATc1 or NFATc2 specific gene silencing in 3 T-ALL GC resistant cell line models and primary cells increases dexamethasone response, by restoring GR canonical transcriptional activity through the increaseexpression of BIM GR targetgene (p value <0.05). Conversely, NFATc1 or NFATc2 overexpression in a murine T-ALL GC sensitive cell line confers resistance to dexamethasone treatment. Interestingly, by Gene Expression Profile (GEP) and Nuclear Magnetic Resonance (NMR) analysis, we observed that NFATc1 gene silencing in GC resistant cells significantly downregulates intracellular cholesterol abundance. Additionally, by Chromatin Immunoprecipitation (ChIP) we revealed that NFATc1 can directly control the transcription of HMGCS1, EBP and DHCR7, key enzymes of cholesterol biosynthesis process (n≥3, p value <0.05 for all the three genes). In agreement, exogenous cholesterol addition to NFATc1 knock-down cell lines rebuild dexamethasone resistance. Conversely, by GEP and flow cytometry analysis we observed that NFATc2 gene silencing in T-ALL GC resistant cells leads to a downregulation of the Wnt/β-catenin signaling pathway and to an increased T-cell differentiation. Furthermore, by ChIP analysis we revealed that in T-ALL GC resistant cells NFATc2 can directly affect the transcription of LRP6, a key Wnt/β-catenin signaling component (n=3, p value <0.05). In agreement, the Wnt/β-catenin signaling activation, by Wnt3a stimulation, restores dexamethasone resistance in NFATc2 knock-down T-ALL cells. Finally, we observed that the inhibition of cholesterol biosynthesis by simvastatin or of Wnt/β-catenin by PRI-724 increases GC sensitivity in T-ALL GC resistant cells by the High-Throughput drug synergism Screening (HTS) and the Highest Single Agent (HSA) approach. Conclusions: Overall, we revealed for the first time the involvement of NFATc1 and NFATc2 transcription factors in supporting GC resistance in T-ALL cells by the modulation of cholesterol biosynthesis and Wnt/β-catenin signaling, both processes well-described in sustaining chemotherapy resistance, paving the rationale to alternative therapeutic options for T-ALL GC resistant pediatric patients.