NOTCH1-induced T-cell Acute Lymphoblastic Leukemia Research Articles

NOTCH1-Induced T-Cell Acute Lymphoblastic Leukemia In Vivo Models.

T-cell acute lymphoblastic leukemia (T-ALL) is primarily a NOTCH1-driven disease, which represents approximately 15% of pediatric and 25% of adult newly diagnosed ALL cases. Gain-of-function NOTCH1 mutations are highly prevalent in T-ALL contributing to almost 60% of the cases. The protocol presented here describes a method for in vivo T-ALL transformation driven by the retroviral transduction of hematopoietic progenitors with oncogenic mutant forms NOTCH1 and subsequent transplant into recipient mice. This T-ALL transformation model allows the interaction between the leukemia cells and the bone marrow microenvironment, better recapitulating the physiological conditions that promote the development of the human disease, providing a versatile tool for both experimental therapeutics and functional genetics studies on T-ALL.

YBX1 as an oncogenic factor in T-cell acute lymphoblastic leukemia.

Y-box-binding protein 1 (YBX1), a member of the RNA-binding protein family, is a critical regulator of cell survival in various solid tumors and acute myeloid leukemia. However, the function of YBX1 in T-cell acute lymphoblastic leukemia (T-ALL) remains elusive. Here, we found that YBX1 was upregulated in patients with T-ALL, T-ALL cell lines, and NOTCH1-induced T-ALL mice. Furthermore, depletion of YBX1 dramatically reduced cell proliferation, induced cell apoptosis, and induced G0/G1 phase arrest invitro. Moreover, YBX1 depletion significantly decreased the leukemia burden in the human T-ALL xenograft and NOTCH1-induced T-ALL mice model invivo. Mechanistically, downregulation of YBX1 markedly inhibited the expression of total AKT serine/threonine kinase (AKT), p-AKT, total extracellular signal-regulated kinase (ERK), and p-ERK in T-ALL cells. Taken together, our results uncovered a critical role of YBX1 in the leukemogenesis of T-ALL, which may have great potential as a biomarker and therapeutic target in T-ALL.

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

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

ASH2L Regulates Genes Involved in the Survival of Leukemic T Cells through a Loci-Specific H3K4me3 Landscape and Leukemogenesis Via Direct Gene Regulation

Acute lymphoblastic leukemia (ALL) is a deadly blood cancer and the most common hematological malignancy in pediatric patients. While treatment response has significantly improved complete remission rates in most patients, the prognosis for children with refractory and relapsed leukemia is bleak, particularly for the subtype T-cell ALL (T-ALL) that exhibits a higher risk for treatment failures and relapses. A greater understanding of T-ALL pathobiology, especially the molecular mechanisms supporting the survival of leukemia-initiating cells (LIC), a leukemic cell subset responsible for treatment failures, is a foundational step toward developing new therapeutic approaches to improve treatment response in patients with high-risk leukemia. While tri-methylation of histone 3 at lysine 4 (H3K4me3) has been shown to support a transcriptional program to preserve leukemia stem cell fate in MLL-associated leukemia, the significance and impact of the H3K4me3 epigenetic landscape in pediatric T-ALL have yet to be established. Because absent, small, or homeotic 2-like protein (ASH2L) is a core component of the mixed-lineage leukemia (MLL) family of histone methyltransferase complex involved in H3K4 methylation, we hypothesized that loss of ASH2L should disrupt the leukemic H3K4me3 landscape impairing the development and maintenance of T-ALL. Here, we show a gradual loss of leukemic T cells in the blood and overall improved survival (p-value < 0.005) in leukemic mice generated by retroviral NOTCH1L1601P-∆P transduction of hematopoietic stem/progenitor cells from mice with conditional Ash2l gene deletion (cKO) in T cells (Ash2Lfl/fl CD4-Cre+). Interestingly, haploinsufficiency and inducible gene deletion in established leukemia using the ROSA-ERCre system also showed improved survival. We found that ASH2L regulates gene expression through H3K4me3 and direct binding to DNA through a combination of RNA-seq, H3K4me3 ChIP-seq, ATAC, and ASH2L ChIP-seq of control (fl/fl) and cKO leukemic T cells (GFP+). Surprisingly, loss of ASH2L caused a gene-specific loss of the H3K4me3 landscape instead of global alterations, suggesting ASH2L regulates the expression of a subset of genes involved in T-ALL maintenance by directing the MLL complex through binding to the nucleosomal DNA. ASH2L regulated genes were further classified based upon alterations in H3K4me3 and ASH2L genome binding as either MLL-dependent or independent. Ash2l cKO NOTCH1-induced T-ALL cells displayed downregulation of gene expression of NOTCH1 and downstream targets (e.g., HES1, DTX1) associated with loss of ASH2L binding to the NOTCH1 gene. Thus, ASH2L regulates the NOTCH1 signaling independently of H3K4me3, which was validated by immunoblots. On the other hand, significant downregulation of the SLIT-ROBO Rho GTPase Activating Protein 3 (SRGAP3) in cKO T-ALL cells was associated with loss of H3K4me3 and ASH2L binding. A comparison of the gene list from mouse T-ALL with patient data sets showed that NOTCH1 and SRGAP3 are downregulated in ASH2L knockout T-ALL cells and healthy individuals compared to T-ALL patients. We are investigating how SRGAP3 regulates the proliferation and survival of leukemic T cells through cell-intrinsic and extrinsic mechanisms. Altogether, ASH2L controls the expression of genes via H3K4me3 (e.g., SRGAP3) and direct gene regulation independently of MLL activity (e.g., NOTCH1). The discovery of ASH2L contribution to T-ALL leukemogenesis will unravel potential targets for treatment.

DT7 peptide-modified lecithin nanoparticles co-loaded with γ-secretase inhibitor and dexamethasone efficiently inhibit T-cell acute lymphoblastic leukemia and reduce gastrointestinal toxicity

T-cell acute lymphoblastic leukemia (T-ALL) is a serious hematologic malignancy and glucocorticoid resistance is the main recurrent cause for a high relapsed and death rate. Here, we proposed an effective therapeutic regimen of combining gamma-secretase inhibitors (GSIs) with dexamethasone (DEX) to overcome glucocorticoid resistance. Moreover, the bone marrow targeting DT7 peptide-modified lecithin nanoparticles co-loaded with DEX and GSI (TLnp/D&G) were developed to enhance T-ALL cells recognition and endocytosis. In vitro cytotoxicity studies showed that TLnp/D&G significantly inhibited cell survival and promoted apoptosis of T-ALL cells. Mechanically, we found that GSIs promoted DEX-induced cell apoptosis by two main synergetic mechanisms: 1) GSIs significantly upregulated glucocorticoid receptor (GR) expression in T-ALL and restored the glucocorticoid-induced pro-apoptotic response. 2) Both DEX and GSI synergistically inhibited BCL2 and suppressed the survival of T-ALL cells. Furthermore, in vivo studies demonstrated that TLnp/D&G showed high bone marrow accumulation and better antileukemic efficacy both in leukemia bearing models and in systemic Notch1-induced T-ALL models, with excellent biosafety and reduced gastrointestinal toxicity. Overall, our study provides new strategies for the treatment of T-ALL and promising bone marrow targeting systems with high transformation potential.

Sirt1 Is a Novel Therapeutic Target in T-ALL

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of T-ALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstratedthe importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in T-ALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemiasleads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Anti-Leukemic Properties of the Kinase Inhibitor OTSSP167 in T Cell Acute Lymphoblastic Leukemia

The remission rates of T-cell acute lymphoblastic leukemia (T-ALL), a hematological malignancy in children, have improved through the years. Despite this advance, 10-20% of the patients do not respond to current therapy, and relapse leukemia is the leading cause of death in children with cancer. Our previous work showed aberrant activation of the actionable MAP2K7-JNK pathway in pediatric T-ALL and proposed this pathway as a target for pharmacological inhibition. Here we demonstrate the anti-leukemic capacity of the kinase inhibitor OTSSP167 in T-ALL via MAP2K7 inhibition. OTSSP167, a MELK inhibitor shown to also inhibit MAP2K7, showed a dose-dependent cytotoxicity in a panel of T-ALL cell lines with IC50 in the nanomolar range (10-50 nM). OTSSP167 induced apoptosis and G2/M cell cycle arrest that correlated with increased cleavage of PARP and caspase 3 and elevated levels of cyclin B1 and phosphorylated CDC2. Immunoblot blot analysis shows OTSSP167 treatment inhibited the MAP2K7-JNK-ATF2 pathway, in addition to MELK, in a panel of T-ALL cell lines. Biochemical kinase assay shows inhibition of MAP2K7 kinase activity with an IC50 of 160 nM. Furthermore, OTSSP167 was able to inhibit MAP2K7 activated by treatment of T-ALL cells with sorbitol. This kinase inhibitor shows low toxicity in mice (10 mg/Kg) and controls leukemia burden in cell-based xenograft model with significant reduction on the expansion of KOPT-K1 cells and improved survival (P=0.003). Furthermore, OTSSP167 was able to control leukemia in patient-derived xenograft (PDX) model and to target leukemia-initiating cells in the NOTCH1-induced T-ALL mouse model. Altogether, OTSSP167 is a potent MAP2K7 inhibitor with low toxicity that emerges as a potential adjuvant drug to increase treatment response and reduce relapses in pediatric T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of the MAP2K7-JNK pathway with 5Z-7-oxozeaenol induces apoptosis in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric leukemia with a worse prognosis than most frequent B-cell ALL due to a high incidence of treatment failures and relapse. Our previous work showed that loss of the pioneer factor KLF4 in a NOTCH1-induced T-ALL mouse model accelerated the development of leukemia through expansion of leukemia-initiating cells and activation of the MAP2K7 pathway. Similarly, epigenetic silencing of the KLF4 gene in children with T-ALL was associated with MAP2K7 activation. Here, we showed the small molecule 5Z-7-oxozeaenol (5Z7O) induces dose-dependent cytotoxicity in a panel of T-ALL cell lines mainly through inhibition of the MAP2K7-JNK pathway, which further validates MAP2K7 as a therapeutic target. Mechanistically, 5Z7O-mediated apoptosis was caused by the downregulation of regulators of the G2/M checkpoint and the inhibition of survival pathways. The anti-leukemic capacity of 5Z7O was evaluated using leukemic cells from two mouse models of T-ALL and patient-derived xenograft cells generated using lymphoblasts from pediatric T-ALL patients. Finally, a combination of 5Z7O with dexamethasone, a drug used in frontline therapy, showed synergistic induction of cytotoxicity. In sum, we report here that MAP2K7 inhibition thwarts survival mechanisms in T-ALL cells and warrants future pre-clinical studies for high-risk and relapsed patients.

A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia.

Long-range oncogenic enhancers play an important role in cancer. Yet, whether similar regulation of tumor suppressor genes is relevant remains unclear. Loss of expression of PTEN is associated with the pathogenesis of various cancers, including T-cell leukemia (T-ALL). Here, we identify a highly conserved distal enhancer (PE) that interacts with the PTEN promoter in multiple hematopoietic populations, including T-cells, and acts as a hub of relevant transcription factors in T-ALL. Consistently, loss of PE leads to reduced PTEN levels in T-ALL cells. Moreover, PE-null mice show reduced Pten levels in thymocytes and accelerated development of NOTCH1-induced T-ALL. Furthermore, secondary loss of PE in established leukemias leads to accelerated progression and a gene expression signature driven by Pten loss. Finally, we uncovered recurrent deletions encompassing PE in T-ALL, which are associated with decreased PTEN levels. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in cancer.

Monocyte-Derived Leukemia-Associated Macrophages Facilitate Extramedullary Distribution of T-cell Acute Lymphoblastic Leukemia Cells.

Macrophages play important roles in both physiologic and pathologic processes and arise from successive waves of embryonic and adult hematopoiesis. Monocyte-derived macrophages (MOMF) exert distinct functions under pathologic conditions, and leukemia-associated macrophages (LAM) show considerable diversities in activation and functional phenotype. However, their origin and pathologic roles have not been well elucidated. Here we used wild-type and CCR2-/- mice to study the pathologic roles of monocyte-derived LAM in extramedullary tissues in models of Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL). MOMF existed in the resting liver and spleen. In the spleen, Ly6C+ monocytes gave rise to the Ly6C+ macrophage subset. Furthermore, an increase of monocyte-derived LAM, including the Ly6C+ subset, was detected in the extramedullary tissues in leukemic mice. More monocyte-derived LAM, including Ly6C+ LAM, was detected in the spleens of leukemic mice transplanted with exogeneous mononuclear cells. Moreover, Ly6C+ LAM exhibited increased M1-related characteristics and contributed to sterile inflammation. In CCR2-/- leukemic mice, reduced Ly6C+ LAM, relieved sterile inflammation, and reduced distribution of leukemia cells were detected in extramedullary tissues. In addition, monocyte-derived Ly6C+ LAM expressed high levels of CCL8 and CCL9/10. Blocking CCR1 and CCR2 relieved hepatosplenomegaly and inhibited the extramedullary distribution of leukemia cells in T-ALL mice. Collectively, our findings reveal the multifaceted pathologic roles of monocyte-derived LAM in T-ALL progression. SIGNIFICANCE: This study links monocyte-derived leukemia-associated macrophages with noninfectious inflammation and extramedullary distribution of leukemia cells during leukemia progression, providing new insight into macrophage-based immunotherapy in leukemia.

Abstract 3434: A tumor suppressor enhancer of PTEN in T-cell development and transformation

Abstract Recent studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL), identifying several oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic lesions in cancer are located in intergenic regions, whose role remains poorly understood. In this context, 20% of T-ALL patients show loss of expression of the tumor suppressor PTEN, but not all can be explained by mutations/deletions of its coding region, defective splicing or promoter hypermethylation. Thus, we hypothesized that alterations of yet unidentified enhancers might play a critical role in the regulation of PTEN expression in T-ALL. To test this, we first performed 4C-seq of the PTEN promoter in human T-ALL cells. Integration of our results with epigenetic profiling revealed a distal non-coding region (named PE for PTEN enhancer) that shows concomitant interaction with the PTEN promoter and bona fide enhancer marks. Moreover, reciprocal 4C-seq assays using PE as viewpoint confirmed its interaction with the PTEN promoter. Next, multispecies DNA sequence alignment revealed notable conservation of PE in mammals and detailed analysis of H3K27ac across 64 cell lines and tissues revealed that PE is specifically active in T-cells and other hematopoietic cells, but is largely repressed in non-hematopoietic cells. Moreover, luciferase assays showed strong, orientation-independent activation of reporter constructs containing either the mouse or human PE sequence in Jurkat cells, and CRISPR/Cas9-induced deletion of PE in human T-ALL cells led to reduced PTEN levels, increased phospho-AKT and enhanced cellular proliferation. To formally test the functional relevance of PE in T-cell development and transformation, we generated PE knockout and conditional knockout mice. PE-null animals are viable and show a marked increase in thymus size and cellularity as their only developmental alteration. Mechanistically, this phenotype is associated with a marked reduction in Pten expression. Next, we analyzed the role of PE in the induction of NOTCH1-driven T-ALLs by transplanting mice with PE wild-type or knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1. In this context, mice transplanted with NOTCH-infected PE-null cells showed 100% penetrance and developed T-ALL ~50 days after transplant. However, mice transplanted with NOTCH-infected PE wild-type cells showed delayed T-ALL kinetics and reduced penetrance. In addition, secondary loss of PE in already established NOTCH1-induced T-ALLs from PE conditional knockout mice led to accelerated T-ALL progression and a gene expression signature driven by Pten loss. Finally, analyses of human T-ALL patient samples uncovered recurrent deletions encompassing PE without concomitant deletion of the PTEN coding sequence. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in the pathogenesis of human leukemia. Citation Format: Luca Tottone, Olga Lancho, Maria Victoria da Silva-Diz, Shirley Luo, Shreeta Chakraborty, Pedro P. Rocha, Daniel Herranz. A tumor suppressor enhancer of PTEN in T-cell development and transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3434.

Phf6 Loss Enhances HSC Self-Renewal Driving Tumor Initiation and Leukemia Stem Cell Activity in T-ALL.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. SIGNIFICANCE: Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL.This article is highlighted in the In This Issue feature, p. 305.

Abstract 49: Synergistic antileukemic therapies in NOTCH1-induced T-ALL

Abstract Over 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL) harbor activating mutations in Notch1 gene, making this receptor a promising therapeutic target for T-ALL. Υ-secretase inhibitors (GSIs), which block a proteolytic cleavage required for NOTCH1 activation, have been clinically explored for the treatment of T-ALL. However, lack of primary responses and relapses have prevented further development of GSIs in the clinic, highlighting the need to identify novel therapies synergistic with NOTCH1 inhibition for the treatment of highly aggressive NOTCH1-mutant T-ALL. To identify new antileukemic drugs for T-ALL we designed an expression-based drug screen utilizing drug profiles contained in the Connectivity Map (cMAP) to search for positive connections with gene signatures driven by NOTCH1 inhibition in this disease. This analyses uncovered 17 compound candidates for synergistic activity with NOTCH1 inhibition, including drugs with known mechanism of action: inhibitors of the mTOR/PI3K/AKT pathway and histone deacetylase inhibitors; as well as compounds with unrelated mechanisms, pyrvinium pamoate (antihelmintic) and withaferin A (antiangiogenic). Pair-wise gene set enrichment analyses on the expression profile driven by each of these drugs in T-ALL cells revealed compounds with transcriptional programs partially overlapping those driven by PI3K/AKT inhibitors, and drugs with distinctive expression profiles, such as withaferin A, suggestive of a unique mode of action. We next characterized the activity of each of the cMAP hits alone and in combination with the GSI DBZ in vitro using a panel of 5 NOTCH1-mutant T-ALL cell lines (CUTLL1, DND41, HPBALL, RPMI and CCRF-CEM). In this analysis, 5 drugs scored as highly active (IC50&amp;lt;0.5) and synergistic (combination index, CI &amp;lt;0.4) in combination with the GSI DBZ: withaferin A, parthenolide, rapamycin, wortmannin, and vorinostat. In addition, 3 out of the 5 drug candidates displayed proapoptotic activity by flow cytometry in cells treated with each of the drugs alone and in combination with DBZ. Among them, withaferin A stood up as the most cytotoxic GSI-synergistic drug candidate. In addition, evaluation of withaferin A activity as single agent and in combination with DBZ in genetic mouse models and primary-derived xenografts of NOTCH1-mutant T-ALL in vivo demonstrated strong antileukemic effects for the combination of GSI and withaferin A. Functional annotation and DeMAND algorithm analyses on the expression profile of withaferin A revealed downregulation of genes and pathways involved in protein translation and identified 21 components of the ribosome and the translation machinery as potential effectors of the antitumoral activity of the drug in T-ALL, including two subunits of the eIF2A translation complex (eIF2S1 and eIF2S2) which controls translation initiation via phosphorylation of the eIF2S1 subunit under conditions of cellular stress. Polysome profiling, nascent protein, and Western blot analyses of cells treated with withaferin A confirmed an inhibitory effect of the drug in protein translation mediated by eIF2S1 phosphorylation. Consistently, expression of a non-phosphorylable mutant form of eIF2S1 in leukemia cell lines rescued the effects of withaferin A in vitro. Our results illustrate the power of our expression-based and in vivo validation approach toward the identification of antitumor drug combinations for the treatment of human cancer. Citation Format: Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Yue Qin, Daniel Herranz, Mukesh Bansal, Tiziana Girardi, Elisabeth Paietta, Martin S. Tallman, Jacob M. Rowe, Kim De Keersmaecker, Andrea Califano, Adolfo Ferrando. Synergistic antileukemic therapies in NOTCH1-induced T-ALL [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 49.

Synergistic antileukemic therapies in NOTCH1-induced T-ALL

The Notch1 gene is a major oncogenic driver and therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL). However, inhibition of NOTCH signaling with γ-secretase inhibitors (GSIs) has shown limited antileukemic activity in clinical trials. Here we performed an expression-based virtual screening to identify highly active antileukemic drugs that synergize with NOTCH1 inhibition in T-ALL. Among these, withaferin A demonstrated the strongest cytotoxic and GSI-synergistic antileukemic effects in vitro and in vivo. Mechanistically, network perturbation analyses showed eIF2A-phosphorylation-mediated inhibition of protein translation as a critical mediator of the antileukemic effects of withaferin A and its interaction with NOTCH1 inhibition. Overall, these results support a role for anti-NOTCH1 therapies and protein translation inhibitor combinations in the treatment of T-ALL.

Inactivation of KLF4 promotes T-cell acute lymphoblastic leukemia and activates the MAP2K7 pathway.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a high incidence of relapse in pediatric ALL. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate the onset of leukemia and worsen disease progression are largely unknown. Here, we show that the gene encoding the transcription factor KLF4 is inactivated by DNA methylation in children with T-ALL. In mice, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL by enhancing the G1-to-S transition in leukemic cells and promoting the expansion of leukemia-initiating cells. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7. Our results showed that in murine and pediatric T-ALL, loss of KLF4 leads to aberrant activation of MAP2K7 and of the downstream effectors JNK and ATF2. As a proof-of-concept for the development of a targeted therapy, administration of JNK inhibitors reduced the expansion of leukemia cells in cell-based and patient-derived xenograft models. Collectively, these data uncover a novel function for KLF4 in regulating the MAP2K7 pathway in T-ALL cells, which can be targeted to eradicate leukemia-initiating cells in T-ALL patients.

ADAR1 Knockout Inhibits Notch1-induced T-ALL in Mice

To investigate the effect of ADAR1 on the occurrence and development of mouse T cell acute lymphoblastic leukemia (T-ALL). Lck-Cre; ADAR1lox/lox mice were generated through interbreeding. The lineage-cells of Lck-Cre; ADAR1lox/lox mice and the control were enriched respectively by the means of MACS, and the lin- cells were transfected with retrovirus carrying MSCV-ICN1-IRES-GFP fusion gene. Then the transfection efficiency was detected by the means of FACS, and the same number of GFP+ cells were transplanted into lethally irradiated recipient mice to observe the survival of mice in 2 recipient group after transplantation. T cell-specific knockout ADAR1 mice were generated, and Notch1-induced T-ALL mouse model was established successfully. The leukemia with T-ALL characteristics occured in the mice of control group, but did not in the ADAR1 kmockout mice after transplantation. ADAR1 plays a key role in the incidence and development of Notch1-induced T-ALL.

CDK6-mediated repression of CD25 is required for induction and maintenance of Notch1-induced T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of acute leukemia, characterized by frequent activation of Notch1 or AKT signaling, where new_therapeutic approaches are needed. We showed previously that Cyclin-dependent kinase 6 (CDK6) is required for thymic lymphoblastic lymphoma induced by activated AKT. Here, we show CDK6 is required for initiation and maintenance of Notch-induced T-ALL. In a mouse retroviral model, hematopoietic stem/progenitor cells lacking CDK6 protein or expressing kinase-inactive (K43M) CDK6 are resistant to induction of T-ALL by activated Notch, whereas those expressing INK4-insensitive (R31C) CDK6 are permissive. Pharmacologic inhibition of CDK6 kinase induces CD25 and RUNX1 expression, cell cycle arrest, and apoptosis in mouse and human T-ALL. Ablation of Cd25 in a K43M background restores Notch-induced T-leukemogenesis, with disease that is resistant to CDK6 inhibitors in vivo. These data support a model whereby CDK6-mediated suppression of CD25 is required for initiation of T-ALL by activated Notch1, and CD25 induction mediates the therapeutic response to CDK6 inhibition in established T-ALL. These results both validate CDK6 as a molecular target for therapy of this subset of T-ALL and suggest that CD25 expression could serve as a biomarker for responsiveness of T-ALL to CDK4/6 inhibitor therapy.

Synergistic Targeting of Protein Translation and Inhibition of NOTCH Signaling in T-ALL

Oncogenic NOTCH signaling is a major driver of T-cell transformation in T-cell acute lymphoblastic leukemia (T-ALL). However, clinical studies testing the efficacy of NOTCH1 inactivation with γ-secretase inhibitors (GSIs) have shown limited antileukemic activity for these drugs as single agents. Here we used an expression-based virtual screening approach and network perturbation analyses to identify and functionally characterize new highly active antileukemic drugs synergistic with NOTCH1 inhibition in T-ALL. Gene expression profiling studies have shown a prominent gene expression signature dominated by genes involved in growth and metabolism downstream of NOTCH1 in T-ALL. Notably, loss of the PTEN tumor suppressor gene confers resistance to GSI therapy and effectively rescues the gene expression signature induced by NOTCH1 inhibition in T-ALL. We hypothesized that drugs inducing transcriptional programs overlapping with those driven by NOTCH1 inhibition and antagonizing those resulting from PTEN loss could have synergistic antileukemic effects with GSIs in PTEN wild type and PTEN null leukemia cells. To address this question we generated gene expression signatures from Pten conditional-inducible knockout NOTCH1-driven leukemias in basal condition, upon NOTCH1 inhibition by GSI treatment and upon deletion of Pten. Connectivity Map (cMAP) analysis in this series identified 17 high scoring compounds as candidate antileukemic drugs (p<0.01). Reassuringly these included two inhibitors of the mTOR/PI3K/AKT pathway (rapamycin, wortmannin), but also histone deacetylase inhibitors (vorinostat, trichostatin A and valproic acid), phenothiazine antipsychotic drugs (trifluoperazine and thioridazine), antimalarial agents (astemizole, mefloquine) and compounds with less characterized activities such as withaferin A, parthenolide and pyrvinium pamoate. Transcriptional profiling followed by pairwise gene set enrichment analysis of these compounds identified groups of drugs with highly interconnected transcriptional programs suggestive of an overlapping mechanism of action (e.g. mTOR/PI3K inhibitors, HDAC inhibitors and phenothiazines), as well as compounds with more unique expression signatures suggestive of a more distinct mode of action (e.g. withaferin A, astemizole and mefloquine). Detailed characterization of the antileukemic effects of these 17 cMAP hits alone and in combination with the GSI DBZ in a broad panel of human NOTCH1-mutated T-ALL cell lines, identified withaferin A, rapamycin, wortmannin, parthenolide and vorinostat as the most active (lethal dose 50 <0.5 µM) and GSI-synergistic (combination index <0.4) drugs in this series. Among these, withaferin A, stood out as the most cytotoxic and GSI-synergistic compound against both PTEN positive and PTEN null T-ALL cell lines. Moreover, withaferin A treatment of primary mouse NOTCH1-induced T-ALLs and primary human T-ALL xenografts demonstrated strong and GSI-synergistic antileukemic activity in vivo.To address the mechanisms mediating the antileukemic effects of withaferin A we performed a detailed analysis of the gene expression signatures induced by this drug in T-ALL lymphoblasts. These studies revealed a strong enrichment of downregulated genes involved in translation regulation in T-ALL cells upon treatment with withaferin A (p<0.001). Mechanistically, transcriptional network perturbation analysis identified the eIF2A translation initiation complex as a potential effector of the antileukemic effects of withaferin A, and withaferin A treatment induced strong dose dependent phosphorylation of eIF2S1 in position S51, a modification responsible for blocking the activity of the eIF2A complex. Consistently, polysome profiling and nascent-protein assays revealed decreased translation in T-ALL cells treated with withaferin A. In this context, expression a phosphomimetic mutant form of eIF2S1 (S51D) impaired leukemia cell viability. Moreover, expression of a non-phosphorylatable form of eIF2S1 (eIF2S1 S51A) in T-ALL cells abrogated the antileukemic effects of withaferin A.These results support a direct role of eIF2S1 phosphorylation and the inhibition of eIF2A-dependent translation as a critical mediators of the antileukemic effects of withaferin A in T-ALL and a role for the combination of GSIs and inhibitors of protein translation for the treatment of high risk T-ALL. DisclosuresCalifano:Therasis Inc: Employment; Cancer Genetics Inc: Consultancy; Ipsen pharmaceuticals: Consultancy; Thermo Fischer Scientific: Consultancy.

Notch1-induced T cell leukemia can be potentiated by microenvironmental cues in the spleen.

BackgroundLeukemia is a systemic malignancy originated from hematopoietic cells. The extracellular environment has great impacts on the survival, proliferation and dissemination of leukemia cells. The spleen is an important organ for extramedullary hematopoiesis and a common infiltration site in lymphoid malignancies. Splenomegaly, frequently observed in T cell acute lymphoblastic leukemia (T-ALL), is associated with poor prognosis. However, how the spleen microenvironment distinctly affects T-ALL cells as opposed to bone marrow (BM) microenvironment has not been addressed.MethodsA Notch1-induced mouse T-ALL model was applied in this study. Flow cytometry and two-photon fluorescence microscopy were used to analyze early distribution of T-ALL cells. MILLIPLEX® MAP Multiplex Immunoassay was performed to measure cytokine/chemokine levels in different microenvironments. Transwell and co-culture experiments were used to test the effects of splenic microenvironment in vitro. Splenectomy was performed to assess the organ specific impact on the survival of T-ALL-bearing mice.ResultsMore leukemia cells were detected in the spleen than in the BM after injection of T-ALL cells by flow cytometry and two-photon fluorescence microscopy analysis. By screening a panel of cytokines/chemokines, a higher level of MIP-3β was found in the splenic microenvironment than BM microenvironment. In vitro transwell experiment further confirmed that MIP-3β recruits T-ALL cells which express a high level of MIP-3β receptor, CCR7. Furthermore, the splenic microenvironment stimulates T-ALL cells to express a higher level of MIP-3β, which further recruits T-ALL cells to the spleen. Co-culture experiment found that the splenic microenvironment more potently stimulated the proliferation and migration of T-ALL cells than BM. Moreover, the mice transplanted with T-ALL cells from the spleen had a shorter life span than those transplanted from BM, suggesting increased potency of the T-ALL cells induced by the splenic microenvironment. In addition, splenectomy prolonged the survival of leukemic mice.ConclusionsOur study demonstrates an organ specific effect on leukemia development. Specifically, T-ALL cells can be potentiated by splenic microenvironment and thus spleen may serve as a target organ for the treatment of some types of leukemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0071-7) contains supplementary material, which is available to authorized users.

A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia.

Efforts to identify and annotate cancer driver genetic lesions have been almost exclusively focused on the analysis of protein coding genes. Here we identify a new long-range acting MYC enhancer controlled by NOTCH1, targeted by recurrent chromosomal duplications in human T-cell acute lymphoblastic leukemia (T-ALL). This highly conserved regulatory element, hereby named N-Me for NOTCH MYC enhancer, is located within a broad super-enhancer region +1.47 Mb from the MYC transcription initiating site, interacts with the MYC proximal promoter and induces orientation-independent MYC expression in reporter assays. Moreover, analysis of N-Me knockout mice demonstrates a selective and essential role of this regulatory element during thymocyte development and in NOTCH1-induced T-ALL. Altogether, these results identify N-Me as a long range oncogenic enhancer directly implicated in the pathogenesis of human leukemia and highlight the fundamental importance of the NOTCH1-MYC regulatory axis in T-cell transformation and as therapeutic target in T-ALL.