Panel Of T-cell Acute Lymphoblastic Leukemia Cell Lines Research Articles

Antileukemic properties of the kinase inhibitor OTSSP167 in T-cell acute lymphoblastic leukemia.

Novel drugs are needed to increase treatment response in children with high-risk T-cell acute lymphoblastic leukemia (T-ALL). Following up on our previous report on the activation of the MAP2K7-JNK pathway in pediatric T-ALL, here we demonstrate that OTSSP167, recently shown to inhibit MAP2K7, has antileukemic capacity in T-ALL. OTSSP167 exhibited dose-dependent cytotoxicity against a panel of T-ALL cell lines with IC50 in the nanomolar range (10-50 nM). OTSSP167 induces apoptosis and cell cycle arrest in T-ALL cell lines, associated at least partially with the inhibition of MAP2K7 kinase activity and lower activation of its downstream substrate, JNK. Other leukemic T-cell survival pathways, such as mTOR and NOTCH1 were also inhibited. Daily intraperitoneal administration of 10 mg/kg OTSSP167 was well tolerated, with mice showing no hematological toxicity, and effective at reducing the expansion of human T-ALL cells in a cell-based xenograft model. The same dosage of OTSSP167 efficiently controlled the leukemia burden in the blood, bone marrow, and spleen of 3 patient-derived xenografts, which resulted in prolonged survival. OTSSP167 exhibited synergistic interactions when combined with dexamethasone, L-asparaginase, vincristine, and etoposide. Our findings reveal novel antileukemic properties of OTSSP167 in T-ALL and support the use of OTSSP167 as an adjuvant drug to increase treatment response and reduce relapses in pediatric T-ALL.

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.

New Drug Repositioning Candidates for T-ALL Identified Via Human/Murine Gene Signature Comparison.

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive subtype of leukemia for which important progress in treatment efficiency have been made in the past decades to reach a cure rate of 75%–80% nowadays. It is nevertheless mandatory to find new targets and active molecules for innovative therapeutic strategies as relapse is associated with a very dismal outcome. We designed an experimental workflow to highlight the conserved core pathways associated with leukemogenesis by confronting the gene expression profiles (GEPs) of human T-ALL cases to the GEP of a murine T-ALL representative model, generated by the conditional deletion of the PTEN tumor suppressor gene in T cell precursors (tPTEN-/-). We identified 844 differentially expressed genes, common GEPs (cGEP) that were conserved between human T-ALL and murine signatures, and also similarly differentially expressed, compared to normal T cells. Using bioinformatic tools we highlighted in cGEPan upregulation of E2F, MYC and mTORC1. Next, using Connectivity Map (CMAP) and CMAPViz a visualization procedure for CMAP data that we developed, we selected in silico three FDA-approved, bioactive molecule candidates: α-estradiol (α-E), nordihydroguaiaretic acid (NDGA) and prochlorperazine dimaleate (PCZ). At a biological level, we showed that the three drugs triggered an apoptotic cell death in a panel of T-ALL cell lines, activated a DNA damage response and interfered with constitutive mTORC1 activation and c-MYC expression. This analysis shows that the investigation of conserved leukemogenesis pathways could be a strategy to reveal new avenues for pharmacological intervention.

Multi-Omic Based Antigen Discovery for the Immunotherapy of Pediatric Acute T Cell Lymphoblastic Leukemia

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease due to treatment related complications and poor prognosis of patients with relapsed disease. Immunotherapy with monoclonal antibodies (MAbs) and/or chimeric antigen receptor (CAR) T-cells for T-ALL is limited by identification of tumor specific target antigens. Differential expression is necessary to prevent on-target/off-tumor toxicities and fratricide of activated T-cells. Targeting multiple antigens can bypass immune escape and result in improved T-cell effector function, since antigen density correlates with T-cell activation. Here we designed a pipeline (Figure 1) to identify unique surface antigens expressed in T-ALL using proteomic and transcriptomic analyses followed by flow cytometry validation, and functional studies with CAR T cells targeting the identified antigens. We generated an Illumina total stranded RNAseq library from healthy donor myeloid and lymphoid cells of bone marrow, peripheral blood and cord blood (N= 116). We compared data to 265 St. Jude pediatric T-ALL samples and against 53 normal tissue expression data from the GTEx (Genotype-Tissue Expression) project. To analyze the T-cell surface proteome, we isolated plasma membrane fractions from 11 samples including healthy T-cells and T-ALL cell lines using a differential centrifugation-based method. The purity of the plasma membrane fraction was confirmed by western blot. Na+/K+ ATPase and GAPDH were used as controls for the plasma membrane and cytosolic fractions respectively. Following plasma membrane enrichment, the membrane proteins were applied for proteomic analysis using an advanced TMT-L/LC-MS/MS pipeline, and the acquired proteomic data were further processed via the JUMP software suite. 997 unique proteins were quantified from the membrane fractions. Integrated analysis the transcriptomic and proteomic datasets showed significant correlation and yielded a list of candidate genes, which were validated by flow cytometry on a panel of T-ALL cell lines (CCRF, RPMI8402, and MOLT3) and resting and activated T-cells from healthy donors. We identified GRP78 as one of the differentially expressed cell surface antigens and further confirmed its expression on additional T-ALL cell lines (KE37, PF382, PEER, CEMC7) and 3 PDX samples. Finally, we generated GRP78-CAR T cells and demonstrate that GRP78-CAR T cells recognize and kill GRP78+ T-ALL cells and have potent antitumor activity in xenograft and PDX models. We have established an unbiased pipeline to identify differentially expressed antigens on the cell surface of T-ALL blasts and created a healthy tissue RNAseq library. The results from our analyses are encouraging and interrogation of our pipeline has yielded differentially expressed immunotherapy targets for the treatment of relapsed refractory T-ALL. Our results highlight the importance of integrated surface proteomics and transcriptomics analysis. Figure 1: Outline of strategy for target selection: Figure Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Gottschalk:Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; TESSA Therapeutics: Other: research collaboration; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Merck and ViraCyte: Consultancy. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties.

Identification of a New Targeted Therapy for T-Cell Acute Lymphoblastic Leukemia and Studies on Its Mechanism of Action

Background: Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy, of which T- cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset. Due to the advent of new therapies, T-ALL now has a 5-year event-free survival (EFS) rate exceeding 85%. However, some patients still relapse and display resistance to therapy. Moreover, adverse side-effects of intensive chemotherapy worsen the duration of treatment. Therefore, we still need to improve our current treatment beyond that of the chemotherapeutic approaches. It has been shown that the maturation stage of T-ALL decides its dependency on Bcl-2/Bcl-xL. The immature early T cell progenitor ALL (ETP-ALL) rely on Bcl-2 for their survival while all the other stages of T-ALL and primary patient samples depend on Bcl-xL. Bcl-2 inhibitors have thus shown to display promising antitumor activity against ETP-ALL, a subgroup with a high risk of relapse, but with a variable response across these patients. Therefore, there is a need for predictive biomarkers and further investigation towards finding a combination of drugs for the treatment of these patients. Methodology & Aim: We screened 10 different T-ALL cell lines with a combination of Bcl-2 inhibitor and a panel of 378 protein kinase inhibitors and identified polo-like kinase inhibitor as a promising candidate. We thus aimed to study the combined effect of Bcl-2 and PLK1 inhibition in a panel of T-ALL cell lines and in a PDX model of chemo-resistant childhood T-ALL. We also investigated the underlying mechanism of drug synergy by various biochemical assays. Results: Cell viability of 14 T-ALL cell lines was determined after being subjected to Bcl-2 inhibitor (ABT-199) and PLK1 inhibitor (BI-6727). All cell lines responded well to BI6727 with an EC50 of less than 70nM. However, they showed differential response to ABT199 with only 3 cell lines being sensitive with an EC50 of less than 40nM. The mRNA levels of Bcl-2, Bcl-xL and PLK 1, 2, 3 and 4 were determined by qRT-PCR. PLK1 was found to be highly expressed in all the cell lines as compared to the rest of the 3 PLK family proteins. ABT-199-sensitive cell lines showed lower Bcl-xL mRNA levels irrespective of their Bcl-2 expression, and displayed synergy with BI-6727. A higher degree of apoptosis was also observed in the combination treatment as compared to a single drug. Immunoblot analysis revealed cleavage of PARP1 and lower levels of c-Myc and MCL1 expression in the presence of both ABT-199 and BI-6727. Conclusions: Upregulation of the anti-apoptotic BCL2 family members is one of the canonical ways for cancer cells to escape apoptosis. In the past years, several highly selective and potent BCL2 inhibitors have been developed and showed promising efficacy in various cancers. We found that the sensitivity of T-ALL cell lines to ABT-199 is largely determined by the lower levels of Bcl-xL expression. Furthermore, ABT-199 displays synergy with the PLK inhibitor. T-ALL cell lines predominantly express PLK1 and thus the combinatorial effect of ABT-199 and BI-6727 is mediated through the pharmacological inhibition of both BCL2 and PLK1. Currently, we are generating iRFP-expressing T-ALL cell lines which will be used to check drug efficacy in vivo. Furthermore, we have collected chemo-resistant PDX cell lines which will be used to verify the cell line data. Besides its role in cell cycle control, we still have very limited knowledge about the function of PLK1 in leukemia. Thus, studying its role in T-ALL cell lines by knocking down PLK1 with CRISPR/Cas9 technology will provide an important insight. Disclosures No relevant conflicts of interest to declare.

Pharmacological Inhibition of MAP2K7 Induces Apoptosis and Cell Cycle Arrest in T-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and relapsed ALL is a leading cause of cancer-related death in children. While advances in frontline therapy have led to an 85% cure rate, relapsed ALL patients face a dismal prognosis necessitating identification of novel targets and development of alternative therapies. Amongst the two sub types of ALL, T-cell acute lymphoblastic leukemia (T-ALL) occurs less frequently but T-ALL patients have worse prognosis and higher rate of relapse. Our group discovered that the transcription factor KLF4 is heavily repressed via DNA methylation coinciding with aberrant activation of MAP2K7 and the MAP2K7 pathway (Shen et al. Leukemia, 2017). Additionally, we demonstrated, as a proof of principle, that pharmacological inhibition of JNK, the only downstream target of MAP2K7, possesses anti-leukemic properties against T-ALL but presented obstacles in terms of low potency and off-target effects. In order to overcome these limitations, we hypothesize that direct pharmacological inhibition of MAP2K7 improve specificity for targeted approach to T-ALL therapeutics. To examine this hypothesis, we explore the anti-leukemic effects of the MAP2K7 pathway inhibitor, 5Z-7-oxozeaenol, against T-ALL cells because it has been shown to covalently react with a unique cysteine-218 in the ATP binding pocket of MAP2K7. We found that T-ALL cell lines exhibit increased cytotoxic sensitivity to 5Z-7-oxozeaenol (IC50 ranging 0.2-1.1 µM) compared to a non-leukemic control (1.5 µM), and more potent than JNK inhibitor IC50 of 5µM. Additionally, 5Z-7-oxozeaenol reduces the amount of phospho-JNK, in a dose dependent manner, indicative of MAP2K7 pathway inhibition. We purified MAP2K7 protein to directly address specificity of kinase inhibition and found that 5Z-7-oxozeaenol inhibits enzymatic activity in vitro of MAP2K7. In addition, 5Z-7-oxozeaenol also inhibited TAK1, which is the MAP3K upstream of MAP2K7. In a panel of T-ALL cell lines, 5Z-7-oxozeaenol treatment induced apoptosis in MOLT3, Jurkat, and KOPTK1 T-ALL cell lines. Although P12-Ichikawa, RPMI-8402, DND-41, and ALL-SIL T-ALL cell lines underwent cell cycle arrest evidenced by a reduction in percentage of S/G2/M phase cells and increase in percentage of G0 phase cells, further increasing 5Z-7-oxozeaenol dosage proved sufficient for induction of apoptosis based on increase of caspase-3 and caspase-7 cleavage. Through reverse-phase protein array analysis we identified reduced expression of several cell cycle regulator proteins, including CDC25C, Cyclin B1, Cyclin D3, and CDC2 resulting from 5Z-7-oxozeaenol treatment. Conversely, we detected increased expression of cleaved caspase-3 and caspase-7, particularly in MOLT3 and Jurkat cell lines treated with MAP2K7 inhibitor. Immunoblot analysis revealed that 5Z-7-oxozeaenol inhibits the MAP2K7 signaling pathway and induces cell cycle arrest and apoptosis in T-ALL cells. Based on these findings, we demonstrated that 5Z-7-oxozeaenol induces cell cycle arrest and apoptosis in T-ALL cells through inhibition of the MAP2K7 pathway, suggesting that MAP2K7 represents a novel pharmacological target for the development of targeted therapy for high-risk patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

Abstract 3005: Whole-genome sequencing identified novel non-coding mutations causal of oncogene activation in T-cell acute lymphoblastic leukemia

Abstract There is growing evidence that non-coding sequences in human genome often function as transcriptional regulatory elements of protein-coding genes. In fact, germline polymorphisms and somatically acquired mutations within regulatory DNA can profoundly alter chromatin structure and modify gene transcription, directly contributing to tumorigenesis. However, there is a paucity of unbiased genome-wide characterization of somatic non-coding mutations in cancer. Using T-cell acute lymphoblastic leukemia (T-ALL) as a model disease, we herein report a systematic interrogation of driver non-coding genomic alterations by paired whole-genome and transcriptome sequencing of 31 children with T-ALL. To identify non-coding mutations with potential regulatory impact in a genome-wide fashion, our analytical pipeline consisted of 3 approaches: 1) the “hotspot analysis” for recurring mutations at the nearby positions, 2) the “regional recurrence analysis” for predefined regulatory regions with significant enrichment of non-coding mutations, 3) the “transcriptional factor analysis” for mutations that potentially result in gain/loss of transcription factor binding sites and alter expression of adjacent genes. Remarkably, T-ALL oncogenes LMO1 and TAL1 emerged as loci with the most significant recurring non-coding mutations. At the LMO1 locus, 3 patients (9.7%) showed an identical single-nucleotide mutation proximal to the transcription start site of the long isoform of LMO1. This recurring mutation resulted in the gain of a canonical Myb binding site (AACGG) and ~120-fold increase in LMO1 transcription compared to patients with wildtype genotype. TAL1 overexpression was observed in 15 patients, of whom 11 had intrachromosomal rearrangement (STIL-TAL1 fusion). The remaining 4 patients had somatic insertion that created a MYB-mediated super enhancer, consistent with recent reports. LMO1 enhancer mutation was further confirmed in an independent validation cohort (N=26), in which we additionally identified a novel intrachromosomal rearrangement between MED17 and LMO1 resulting in transcriptional activation of the latter. In a panel of T-ALL cell lines, LMO1 enhancer mutation was again associated with dramatic overexpression of LMO1, an active enhancer histone mark (H3K27ac), Dnase hypersensitivity, and allele-specific binding of MYB. Interestingly, we also observed robust binding of TAL1, CREBBP, RUNX1, ETS1, ELF1 and RNA Polymerase II at this site. Reporter gene assay confirmed the MYB-mediated transcription activation effects of this LMO1 enhancer mutation in vitro. In this genome-wide investigation of non-coding mutations in T-ALL, we identified novel enhancer mutations with drastic effects on oncogene activation. Our findings expand the understanding of how genomic alterations in regulatory DNA contribute to cancer pathogenesis. Citation Format: Maoxiang Qian, Shaoyan Hu, Hui Zhang, Yu Guo, Jin Yang, Xujie Zhao, Lin Wan, Jun Lu, Jian Pan, Meimei Chang, Shirley K. Kham, Yong Cheng, Chunliang Li, Allen E. Yeoh, Anders Skanderup, Jun J. Yang. Whole-genome sequencing identified novel non-coding mutations causal of oncogene activation in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3005. doi:10.1158/1538-7445.AM2017-3005

Abstract B25: BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia

Abstract Introduction: Despite the improved survival of patients with T-cell acute lymphoblastic leukemia (T-ALL), therapy-resistant or refractory T-ALL remains a major clinical challenge. Recently, we and others reported promising therapeutic activity for ABT-199, a highly specific inhibitor of the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), especially in immature T-ALL. Nevertheless, the occurrence of dose-limiting toxicities and the emergence of therapy resistance provides a rationale for the evaluation of ABT-199 as part of combination therapies. Indeed, previous studies have shown that ABT-199 can synergize with conventional chemotherapeutic agents in human T-ALL. BRD4 is an important bromodomain and extra-terminal (BET) protein that facilitates the recruitment of the transcriptional apparatus to acetylated chromatin. Small molecule inhibitors that target BET-bromodomain proteins, such as JQ1, can broadly affect oncogenic gene transcription and have recently emerged as powerful anti-cancer agents in a variety of hematological malignancies, including T-ALL. Here, we evaluated JQ1 and ABT-199 combination therapy and provide in vitro and in vivo evidence that combined BCL-2 and BRD4 inhibition synergistically affects leukemic tumor growth in T-ALL. Experimental Procedures: In vitro synergism between ABT-199 and JQ1 in a panel of T-ALL cell lines was determined by the combination index (CI) based on cell viability 48h after compound administration. For in vivo evaluation of the ABT-199/JQ1 combination therapy, luciferase-labeled T-ALL cells were administered by tail vein injection in immune compromised NOD scid gamma mice. Upon engraftment of leukemic cells, mice were divided into four groups and treated daily with 50mg ABT-199/kg body weight and/or 50mg JQ1/kg body weight for 13 days. The percentage of leukemic cells in the blood and bone marrow after treatment was analyzed using FACS analysis upon staining with an anti-human CD45 antibody. Results: In vitro evaluation of ABT-199 and JQ1 combination therapy revealed strong synergism (CI<0.5) in the T-ALL cell lines LOUCY, DND-41, ALL-SIL and TALL-1. In contrast, no or only slight synergistic effects (CI>0.5) were observed in other T-ALL lines, including KOPTK1, JURKAT, PEER and HPB-ALL. Notably, the degree of synergism was not defined by the IC50 values for ABT-199 or JQ1, but was significantly correlated with BCL-2 expression levels. Next, in vivo drug treatment experiments using luciferase-positive LOUCY T-ALL cells confirmed the synergistic activity of the JQ1/ABT-199 combination therapy as evaluated by bioluminescence, percentage of leukemic blasts in blood or bone marrow and spleen size. Interestingly, although ABT-199 strongly reduced the leukemic burden during the first days of treatment, the therapeutic response of single ABT-199 therapy was not durable. In contrast, JQ1/ABT-199 combination therapy resulted in a sustained therapeutic response associated with tumor regression. As expected, combination therapy resulted in enhanced levels of cleaved PARP and increased caspase activity in synergistic T-ALL cell lines, in line with induction of cellular apoptosis upon ABT-199/JQ1 treatment. To better understand the molecular mechanism by which JQ1 enhances the anti-leukemic properties of ABT-199, we defined its effect on gene expression in T-ALL cell lines. Notably, this analysis showed that the balance of pro- and anti-apoptotic BCL2 family factors was affected by JQ1 treatment. For example, JQ1 treatment caused a significant reduction in BCL2 levels, but also induced a concomitant increase in anti-apoptotic MCL1. In addition, pro-apoptotic factors, such as BIM, were also upregulated upon JQ1 treatment. Conclusions: Here, we show that BET-bromodomain inhibition can sensitize BCL2-positive human T-ALL to ABT-199 treatment by interfering with the balance of pro- and anti-apoptotic BCL2 family members. Citation Format: Sofie Peirs, Filip Matthijssens, Tim Pieters, Niels Vandamme, Geert Berx, Bruce Poppe, Pieter Van Vlierberghe. BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B25.

Abstract B39: BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia

Abstract Introduction: Despite the improved survival of patients with T-cell acute lymphoblastic leukemia (T-ALL), therapy-resistant or refractory T-ALL remains a major clinical challenge. Recently, we and others reported promising therapeutic activity for ABT-199, a highly specific inhibitor of the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), especially in immature T-ALL. Nevertheless, the occurrence of dose-limiting toxicities and the emergence of therapy resistance provides a rationale for the evaluation of ABT-199 as part of combination therapies. Indeed, previous studies have shown that ABT-199 can synergize with conventional chemotherapeutic agents in human T-ALL. BRD4 is an important bromodomain and extra-terminal (BET) protein that facilitates the recruitment of the transcriptional apparatus to acetylated chromatin. Small molecule inhibitors that target BET-bromodomain proteins, such as JQ1, can broadly affect oncogenic gene transcription and have recently emerged as powerful anti-cancer agents in a variety of hematological malignancies, including T-ALL. Here, we evaluated JQ1 and ABT-199 combination therapy and provide in vitro and in vivo evidence that combined BCL-2 and BRD4 inhibition synergistically affects leukemic tumor growth in T-ALL. Experimental Procedures: In vitro synergism between ABT-199 and JQ1 in a panel of T-ALL cell lines was determined by the combination index (CI) based on cell viability 48h after compound administration. For in vivo evaluation of the ABT-199/JQ1 combination therapy, luciferase-labeled T-ALL cells were administered by tail vein injection in immune compromised NOD scid gamma mice. Upon engraftment of leukemic cells, mice were divided into four groups and treated daily with 50mg ABT-199/kg body weight and/or 50mg JQ1/kg body weight for 13 days. The percentage of leukemic cells in the blood and bone marrow after treatment was analyzed using FACS analysis upon staining with an anti-human CD45 antibody. Results: In vitro evaluation of ABT-199 and JQ1 combination therapy revealed strong synergism (CI<0.5) in the T-ALL cell lines LOUCY, DND-41, ALL-SIL and TALL-1. In contrast, no or only slight synergistic effects (CI>0.5) were observed in other T-ALL lines, including KOPTK1, JURKAT, PEER and HPB-ALL. Notably, the degree of synergism was not defined by the IC50 values for ABT-199 or JQ1, but was significantly correlated with BCL-2 expression levels. Next, in vivo drug treatment experiments using luciferase-positive LOUCY T-ALL cells confirmed the synergistic activity of the JQ1/ABT-199 combination therapy as evaluated by bioluminescence, percentage of leukemic blasts in blood or bone marrow and spleen size. Interestingly, although ABT-199 strongly reduced the leukemic burden during the first days of treatment, the therapeutic response of single ABT-199 therapy was not durable. In contrast, JQ1/ABT-199 combination therapy resulted in a sustained therapeutic response associated with tumor regression. As expected, combination therapy resulted in enhanced levels of cleaved PARP and increased caspase activity in synergistic T-ALL cell lines, in line with induction of cellular apoptosis upon ABT-199/JQ1 treatment. To better understand the molecular mechanism by which JQ1 enhances the anti-leukemic properties of ABT-199, we defined its effect on gene expression in T-ALL cell lines. Notably, this analysis showed that the balance of pro- and anti-apoptotic BCL2 family factors was affected by JQ1 treatment. For example, JQ1 treatment caused a significant reduction in BCL2 levels, but also induced a concomitant increase in anti-apoptotic MCL1. In addition, pro-apoptotic factors, such as BIM, were also upregulated upon JQ1 treatment. Conclusions: Here, we show that BET-bromodomain inhibition can sensitize BCL2-positive human T-ALL to ABT-199 treatment by interfering with the balance of pro- and anti-apoptotic BCL2 family members. Citation Format: Sofie Peirs, Filip Matthijssens, Tim Pieters, Niels Vandamme, Geert Berx, Bruce Poppe, Pieter Van Vlierberghe. BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B39.

Synergistic Drug Combinations with a CDK4/6 Inhibitor in T-Cell Acute Lymphoblastic Leukemia

While significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), approximately 10-20% of newly diagnosed patients will experience either induction failure or relapse. Additionally, fewer than 50% of T-ALL patients who experience a relapse are long-term survivors. New targeted therapies are needed for the treatment of this disease.Multiple lines of evidence point to Cyclin D3/CDK4/6 as a potential therapeutic target in T-ALL. Cyclin D3 (CCND3), a direct target of activated NOTCH1, is upregulated in T-ALL, and CCND3 null animals are refractory to NOTCH1 driven T-ALL. CCND3 binds and activates CDK4/6, and the CCND3-CDK complex phosphorylates the tumor suppressor RB leading to cell cycle progression. Previous studies have demonstrated that CDK4/6 small-molecule inhibition is an effective therapeutic strategy for the treatment of NOTCH1-driven T-ALL mouse models.Using the publicly available Genomics of Drug Sensitivity in Cancer data set, we identified NOTCH1 mutations as a biomarker of response and RB mutations as a biomarker of resistance to the CDK4/6 inhibitor palbociclib. We validated that RB null status predicts resistance to the Novartis CDK4/6 inhibitor LEE011 in a panel of T-ALL cell lines. Interestingly, we identified both NOTCH1 mutant, as well as NOTCH1 wildtype, T-ALL cell lines that were sensitive to LEE011 treatment. Mining of publicly available data revealed that CDK6 is consistently marked by a super-enhancer in T-ALL cell lines, both NOTCH1 mutant and wildtype, suggesting another potential reason for sensitivity to CDK4/6 inhibition in this lineage. Treatment with LEE011 also led to a dose-dependent cell cycle arrest and cell death in T-ALL cells, including MOLT4 (NOTCH1 mutant) and MOLT16 (NOTCH1 wildtype).Combinations of drugs with CDK4/6 inhibitors will likely be critical for the successful translation of this drug class because they generally do not induce cell death. Combinations with cytotoxic chemotherapy are predicted to be antagonistic, however, as most of these drugs rely on rapidly proliferating cells, and CDK4/6 inhibition induces cell cycle arrest. To discover effective, and immediately translatable combination therapies with LEE011 in T-ALL, we performed combination studies of LEE011 with agents standardly used for T-ALL treatment, including corticosteroids, methotrexate, mercaptopurine, asparaginase, vincristine and doxorubicin. Combinations of LEE011 with methotrexate, mercaptopurine, vincristine or asparaginase were antagonistic in T-ALL cell lines while the combination with doxorubicin was additive. Combination treatment of LEE011 with corticosteroids had a synergistic effect on cell viability in MOLT4 and MOLT16 cell lines as measured by excess over Bliss additive and isobologram analyses. This combination also decreased phospho RB signaling, increased cell cycle arrest and induced cell death to a greater degree than either drug alone. LEE011 treatment increased CCND3 protein levels, an effect mitigated by glucocorticoid treatment, one possible mechanism contributing to the observed synergy. Additionally, the combination of LEE011 with everolimus, an mTOR inhibitor, was synergistic in these cell lines.We next extended testing to in vivo models of T-ALL. In a MOLT16 orthotopic mouse model, the combination of LEE011 and everolimus significantly prolonged mouse survival compared to treatment with each individual drug alone. The combination of LEE011 with dexamethasone did not extend survival over treatment with LEE011 alone and dexamethasone was inactive in vivo. Both LEE011 and everolimus had on-target activity in the treated mice as measured by inhibition of peripheral blood phospho-RB and phospho-4EBP1. We then tested the combination of LEE011 with dexamethasone in a second mouse model, a MOLT4 orthotopic model. Here, the combination of LEE011 with dexamethasone was more effective in prolonging survival compared to each treatment alone, supporting a heterogeneous response to the combination of LEE011 with dexamethasone in vivo.We conclude that LEE011 is active in T-ALL, and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation in the clinical setting. DisclosuresKim:Novartis Pharmaceuticals: Employment. Stegmaier:Novartis Pharmaceuticals: Consultancy.

The CXCR4/CXCL12 Axis Mediates Chemotaxis, Survival, and Chemoresistance in T-Cell Acute Lymphoblastic Leukemia

Background: While multi-agent chemotherapy has led to remarkable improvements in survival for children with T-cell acute lymphoblastic leukemia (T-ALL), outcomes remain dismal for the ~ 20% of patients that fail therapy. The goal of this study was to elucidate biological mechanisms contributing to chemoresistance in T-ALL. We hypothesized that chemoresistance owes not only to the aberrant signaling pathways intrinsic to the leukemia cells but also to contributions from an abnormal microenvironment in which they reside. The chemokine receptor ligand pair CXCR4/CXCL12 is important for normal leukocyte trafficking and deregulation is frequently observed in several hematologic malignancies. However, the role this axis plays in T-ALL is largely unknown. Here, we test the hypothesis that CXCR4/CXCL12 creates a sanctuary microenvironment that promotes T-ALL survival and provides protection from chemotherapy.Methods: A panel of T-ALL cell lines and primary patient samples expanded in NOD/SCID/c null (NSG) mice were cultured in the presence or absence of the CXCR4 antagonist, AMD3100, with or without chemotherapy. The human bone marrow stromal cell line, HS27a, which constitutively expresses the CXCR4 ligand CXCL12, was used to recapitulate the tumor microenvironment ex vivo. Transwell migration and modified pseudo-emperipolesis assays were used to examine CXCL12-mediated chemotaxis. Multi-parameter flow cytometry was used to evaluate the impact of activation of the CXCR4/CXCL12 axis on signaling networks, cell survival, and chemotherapy resistance.Results: We found CXCR4 membrane expression on all T-ALL cell lines and xenografts samples tested. Incubation with the CXCR4 ligand CXCL12 resulted in activation of survival signaling cascades (PI3K/AKT and MAPK), an effect blocked with AMD3100. Using a transwell system, we found dose dependent chemotaxis of T-ALL lymphoblasts to CXCL12 that was prevented by AMD3100. T-ALL cells also migrated into HS27a stromal cells in a CXCR4/CXCL12 dependent fashion. In addition, co-culturing T-ALL xenografts cells with HS27a imparted a survival advantage that was promptly eliminated by AMD3100 exposure. To test whether the CXCR4/CXCL12 microenvironment contributed to chemotherapy resistance, T-ALL xenografts cells were co-cultured ex vivo in the presence or absence of HS27a stromal cells and bortezomib or dexamethasone. Stromal cells conferred a marked chemoprotective effect that was specifically blocked by AMD3100. This highlights that stroma-mediated chemoresistance in xenograft samples is in part due to stromal-cell mediated activation of the CXCR4/CXCL12 axis.Conclusions: This study provides evidence for a T-ALL microenvironment that exploits the CXCR4/CXCL12 axis for leukemic cell recruitment, enhanced cell survival, and chemotherapy protection. Our findings also implicate the stroma as a major contributor to chemotherapy resistance in primary expanded patient samples partially due to the activation of the CXCR4/CXCL12 axis. These results further suggest that targeting the stroma through inhibition of this axis may be of therapeutic benefit in patients with chemotherapy resistant T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Selectively Targeting Mutated NOTCH1 with a Folate-Thapsigargin Derivative

Gain-of-function mutations in NOTCH1, encoding a trans-membrane receptor involved in a transcriptional activation complex, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Thus, targeting NOTCH1 represents one promising therapeutic opportunity in T-ALL. The implementation of NOTCH1 pathway antagonists, however, poses several challenges secondary to concerns about inhibiting normal Notch receptors, leading to gut toxicity and secondary malignancies.We recently reported the results of a gene-expression based, NOTCH1 chemical genomic loss-of-function screen and a cell-based screen for cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. SERCA (Sarco-endoplasmic reticulum Ca2+ ATPases) emerged at the intersection of these complementary approaches. We discovered that SERCA inhibition impairs the maturation of mutated NOTCH1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human T-ALL cells. A small-molecule SERCA inhibitor, thapsigargin, a sesquiterpene-g-lactone, had on-target activity in mouse models of human T-ALL and also interfered with Notch signaling in Drosophila. Remarkably, thapsigargin preferentially inhibited mutated NOTCH1 receptors. This selectivity provides a therapeutic window not observed with other Notch pathway inhibitors, such as gamma-secretase inhibitors, that are equipotent inhibitors of mutated and wild-type receptors.While thapsigargin is a highly potent tool compound, in its native form, it is unlikely to have direct clinical application because of its global effects on intracellular Ca2+. Aberrant expression of folate receptors (FR) on the surface of cancer cells, including leukemia, has previously been reported, and efforts have been undertaken to leverage folate receptor-mediated endocytosis to deliver cancer therapeutics in vivo. We therefore developed a thapsigargin derivative conjugated with folic acid (JQ-FT) for preclinical testing in T-ALL.The FR gene family includes four members: FOLR1-4. The high affinity of FOLR1 and FOLR2 for folate binding, their endocytic capacity and their restricted expression pattern have prompted evaluation of folate-drug conjugates as a therapeutic approach in cancer. Analysis of gene-expression profiling suggested that FOLR2 is more highly and consistently expressed compared to FOLR1 in human T-ALL cell lines. We confirmed this finding by qRT-PCR across a panel of T-ALL cell lines and three primary patient samples. Surface expression of FOLR1 and FOLR2 measured by flow cytometry paralleled the transcriptional findings. We next validated the dependency of the uptake of a folate-FITC probe in T-ALL cells on active endocytosis via FR. First, we demonstrated that treatment of T-ALL cells with increasing concentrations of folate-FITC corresponds to an increase in measurable fluorescent signal by flow cytometry. The uptake is specific for FR as overexpression of FOLR1 or FOLR2 leads to increased, and folic acid competition decreased, fluorescent signal. Moreover, active endocytosis is supported by data demonstrating that folate-FITC fluorescence is not eliminated with an acidic wash step and that cold temperature does diminish internalization of the chemical probe.Next we demonstrated that increasing concentrations of JQ-FT impede the growth of T-ALL cells and induce apoptosis as measured by the activation of caspase 2. Moreover, the overexpression of ICN1, the intracellular active form of NOTCH1, partially rescues the small-molecule anti-proliferative effect indicating that the JQ-FT retains the on target activity similar to the underivatized thapsigargin. As observed with thapsigargin treatment of T-ALL cells, JQ-FT treatment decreased the levels of the intracellular active form of NOTCH1, ICN1, and the furin-processed transmembrane form of NOTCH1, while the full length precursor accumulated.In summary, these pre-clinical studies confirm the selective uptake of a folate-conjugated thapsigargin by an FR-mediated mechanism with the suppression of mutated NOTCH1 maturation and support the ongoing testing of this approach in mouse models of NOTCH1-mutated T-ALL. If successful, this approach would enhance the therapeutic window of thapsigargin as a NOTCH1 inhibitor providing dual selectivity: leukemia over normal cell and NOTCH1 mutated over wild-type receptors. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic targeting of Polo-like kinase-1 and Aurora kinases in T-cell acute lymphoblastic leukemia

Polo-like kinases (PLKs) and Aurora kinases (AKs) act as key cell cycle regulators in healthy human cells. In cancer, these protein kinases are often overexpressed and dysregulated, thus contributing to uncontrolled cell proliferation and growth. T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous malignancy arising in the thymus from T-cell progenitors. Primary chemoresistant and relapsed T-ALL patients have yet a poor outcome, therefore novel therapies, targeting signaling pathways important for leukemic cell proliferation, are required. Here, we demonstrate the potential therapeutic effects of BI6727, MK-5108, and GSK1070916, three selective inhibitors of PLK1, AK-A, and AK-B/C, respectively, in a panel of T-ALL cell lines and primary cells from T-ALL patients. The drugs were both cytostatic and cytotoxic to T-ALL cells by inducing G2/M-phase arrest and apoptosis. The drugs retained part of their pro-apoptotic activity in the presence of MS-5 bone marrow stromal cells. Moreover, we document for the first time that BI6727 perturbed both the PI3K/Akt/mTORC2 and the MEK/ERK/mTORC1 signaling pathways, and that a combination of BI6727 with specific inhibitors of the aforementioned pathways (MK-2206, CCI-779) displayed significantly synergistic cytotoxic effects. Taken together, our findings indicate that PLK1 and AK inhibitors display the potential for being employed in innovative therapeutic strategies for improving T-ALL patient outcome.

Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia

Constitutively active phosphoinositide 3-kinase (PI3K) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival and drug resistance. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G2/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose- and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway.

The Order of Exposure to Cytotoxic Chemotherapy and the Proteasome Inhibitor Bortezomib Dictates the Extent of Reversal of Chemotherapy Resistance in T-ALL

AbstractAbstract 1355 Introduction:While most children with T-cell acute lymphoblastic leukemia (T-ALL) are cured, the outcome for children who respond poorly to therapy or relapse is almost universally fatal. Our laboratory is interested in understanding the mechanisms contributing to therapy resistance and has developed a mutliparameter phosphoflow cytometry assay to study signal transduction events at a single cell level.Nuclear Factor kappa B (NF-κB) is a family of transcription factors that plays an important role in cancer development by preventing apoptosis and facilitating tumor cell growth. Consistent with this, constitutive NF-κB activity has been reported in T-ALL, and has been suggested as a predictor of relapse. We hypothesized that the proteasome inhibitor Bortezomib, which blocks NF-κB activation, induces apoptosis in T-ALL. We further hypothesized that combining Bortezomib with conventional chemotherapeutic agents sensitizes T-ALL cells to apoptosis and overcomes chemotherapy resistance by altering the balance of downstream pro- and anti- apoptotic mediators. Methods:A panel of T-ALL cell lines was cultured in the presence of vehicle alone, conventional cytotoxic drugs (Dexmethasone, Etoposide, Cyclophosphamide, Cytarabine), Bortezomib, or combinations of these. Cytotoxic effect was measured by trypan blue staining and flow cytometric detection of cleaved caspase-3. Multiparameter phosphoflow cytometry was used to measure expression levels of downstream apoptosis mediators. An advantage of multiparameter phosphoflow cytometry is the ability to gate on caspase 3-negative (i.e., chemotherapy resistant) cells, allowing us to interrogate potential mechanisms of resistance by comparing expression profiles of cell survival proteins before and after treatment. Results:T-ALL cells were exposed to cytotoxic chemotherapy or vehicle for 24 to 48 hours and subjected to phosphoflow cytometry. Analysis of caspase-3 negative cells indicated that chemotherapy resistant cells rewired their signaling networks by upregulating their MAPK, NF-κB, and AKT/PI3K/S6 signaling networks in response to genotoxic stress. This correlated with upregulation of the pro-survival protein survivin, suggesting a potential mechanism for chemotherapy resistance. To test whether Bortezomib could reverse this resistance, cells were exposed to subtoxic concentrations of Bortezomib alone or in combination with cytotoxic drugs. Combination therapy produced only a modest synergistic effect. We then wondered whether the sequence of administration of cytotoxic drug and Bortezomib could potentiate this effect. T-ALL cell lines were therefore 1) pretreated with Bortezomib for 24 hours followed by cytotoxic drug for an additional 24 hours, 2) pretreated with cytotoxic drug for 24 hours followed by addition of Bortezomib for an additional 24 hours, or 3) treated with both agents simultaneously. Surprisingly, the most pronounced cytotoxic synergy was observed when T-ALL cells were pretreated with cytotoxic drug followed by Bortezomib. While simultaneous or pretreatment with Bortezomib had no effect on upregulation of surivin in response to cytoxic chemotherapy, addition of Bortezomib post genotoxic stress resulted in markedly decreased levels of survivin. Conclusions:We found that Bortezomib lowered the apoptotic threshold to conventional cytotoxic drugs and can reverse drug resistance in T-ALL cell lines. Interestingly, the order of exposure to cytotoxic chemotherapy and Bortezomib determines the extent of reversal of chemotherapy resistance. Our data is in agreement with studies in multiple myeloma and provides evidence supporting the use of Bortezomib in combination with conventional cytotoxic agents in the treatment of relapsed or refractory disease. Disclosures:No relevant conflicts of interest to declare.

Activity of the Novel Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 against T-Cell Acute Lymphoblastic Leukemia

Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival, and drug resistance. These observations lend compelling weight to the application of PI3K/Akt/mTOR inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. NVP-BEZ235 was cytotoxic to a panel of T-ALL cell lines as determined by MTT assays. NVP-BEZ235 treatment resulted in cell cycle arrest and apoptosis. Western blots showed a dose- and time-dependent dephosphorylation of Akt and mTORC1 downstream targets in response to NVP-BEZ235. Remarkably, NVP-BEZ235 targeted the side population of both T-ALL cell lines and patient lymphoblasts, which might correspond to leukemia-initiating cells, and synergized with chemotherapeutic agents (cyclophosphamide, cytarabine, dexamethasone) currently used for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic to T-ALL patient lymphoblasts displaying pathway activation, where the drug dephosphorylated eukaryotic initiation factor 4E-binding protein 1, at variance with rapamycin. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment of those T-ALLs that have aberrant upregulation of this signaling pathway for their proliferation and survival.

Direct Inhibition of the Notch Transactivation Complex with Synthetic Constrained Peptides in T-Cell Acute Lymphoblastic Leukemia.

Notch signaling represents a central pathway regulating hematopoiesis, stem cell differentiation, and malignant transformation in human cancer. Activation of highly conserved Notch1 receptors results in cleavage and release of an intracellular domain (ICN1). Following translocation to the nucleus, ICN1 forms a ternary complex with the transcriptional repressor CSL (CBF-1, Suppressor of Hairless and Lag-1) bound to cognate DNA. This event triggers a repressor-to-activator switch, as an interfacial groove is formed which recruits the Mastermind-Like (MAML1) co-activator protein. Activating mutations in NOTCH1 are found in more than 50% of patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL), promoting protein stability and establishing a direct link to disease pathogenesis. Pharmacologic efforts to target the Notch pathway in T-ALL have been directed at gamma secretase, a regulatory enzyme in Notch activation. Recently, the observed clinical resistance to gamma secretase inhibitors has been explained, in part, by additional mutations in the Notch-targeting ubiquitin ligase, Fbxw7, which further increases oncoprotein stability. Therefore, direct inhibitors of ICN1 function are highly desirable. Drawing upon insights afforded by the resolved crystal structure of the DNA-bound ICN1:MAML1:CSL complex, we synthesized a series of hydrocarbon stapled alpha-helical peptides targeting Notch (SAHNs) based on minimal motifs of the MAML protein predicted to engage the composite ICN1:CSL interface. Direct, high-affinity binding to purified components of the Notch complex was confirmed using surface plasmon resonance (SPR). Nuclear access of SAHN1 was confirmed using quantitative epifluorescent and confocal microscopy. Intracellular association with ICN1 and CSL was established using bidirectional affinity chromatography. Using a novel CSL-responsive reporter construct, we observed inhibition of endogenous Notch transactivation by SAHN1 in T-ALL cell lines. Furthermore, SAHN1 induces a dose-dependent knockdown of endogenous Notch1 target genes including HES1, HEY1 and cMYC in T-ALL cell lines. Remarkably, inhibition of Notch signaling by SAHN1 confers selective cytotoxicity at 48 hours in a panel of T-ALL cell lines with known mutations in NOTCH, including those resistant to gamma secretase inhibitors. Supporting an on-target mechanism of action, we have prepared a damaged analogue of SAHN1 containing a two-residue rearrangement (SAHN1D). SAHN1D possesses reduced binding affinity for the Notch complex and despite comparable intracellular access, SAHN1D lacks both transcriptional and cytotoxic effects on cultured T-ALL cell lines in vitro. Efficacy studies have also been performed in vivo using a novel murine model of T-ALL. In summary, we report here the design, biochemical characterization and translational rationale supporting the first direct inhibitor of the Notch transactivation complex in T-ALL.

Activation of the JNK Pathway Mediates Chemoresistance in T-Cell Acute Lymphoblastic Leukemia by Phosphorylation and Proteosomal Degradation of BimEL.

Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.