Human Acute Lymphoblastic Leukemia Research Articles

Cytotoxic Oxygenated Steroids from the Soft Coral Nephthea erecta.

A new 10-demethylated steroid, nephtheasteroid A (1), a new 19-oxygenated steroid, nephtheasteroid B (2) as well as five known steroids 3-7 were isolated from the organic extract of a Taiwanese soft coral Nephthea erecta. The structure was determined by means of IR, MS, and NMR techniques. Among these metabolites, 1 is rarely found in steroids possessing a 19-norergostane skeleton. In vitro cytotoxicity study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that compounds 3 and 4 exhibited cytotoxicity against human chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (Molt-4), human T lymphoblastoid (Sup-T1), and human leukemic monocyte lymphoma (U937), with IC50 of 6.5-14.0 µM.

Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway and NUP214-ABL1 fusion protein in human acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a neoplasm of precursor cells committed to the B-cell and T-cell lineages involving bone marrow and blood, with a rapid onset and frequent chemotherapy resistance and refractory relapses (1). Philadelphia chromosome-positive (Ph+) ALL accounts for 25–30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes, among which BCR-ABL1 is the most commonly found, are markers of very poor prognosis. Amplification of the NUP214-ABL1 oncogene can be detected only in patients with T-ALL (2). The PI3K/Akt/mTOR signaling pathway is activated in many solid cancers and in leukemias and plays a crucial role in tumorigenesis. Furthermore, the presence of RTKs (Receptor Tyrosine Kinases) by ABL1 fusion proteins may result in activation of the PI3K/Akt/mTOR axis. T cell malignancies bearing the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been found. In this work we analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on two NUP214-ABL1 positive T-ALL cell lines, ALL-SIL and PEER that also displayed Akt hyperactivation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis as deducted by a readout of drug efficacy, four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226 (BGT226), ZSTK474 and Torin-2, showed a relevant cytotoxic efficacy on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of the compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability which was associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL.

Molecular Cytotoxic Mechanisms of 1-(3,4,5-Trihydroxyphenyl)-dodecylbenzoate in Human Leukemia Cell Lines.

Recent studies have shown that gallic acid and its alkylesters induce apoptosis in different cell lines. Since new compounds with biological activity and less cytotoxicity to normal cells are necessary for cancer therapy, the aim of this study was to evaluate the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. The cell viability was determined by MTT method. The apoptosis induction was assessed by bromide and acridine orange staining and by Annexin V-FITC Apoptosis Detection kit. The cell cycle analysis was carried out by flow cytometry using propidium iodide. Cytometric analysis was also performed to evaluate the expression of the following proteins: AIF, p53, Bcl-2 and Bax. The mitochondrial potential was also assessed by flow cytometry using MitoView633 kit. The results showed that the compound significantly reduced the cell viability of K562 and Jurkat cells in a concentration and time dependent manner (IC50 of 30 μM). The compound induced cell cycle arrest in G0/G1phase and significantly increased the proportion of cells in the sub-G0/G1phase. Apoptosis was confirmed by the sight of morphological characteristics of apoptosis and by phosphatidylserine externalization (73.47±5.71% of cells expressing annexin). The results also showed that the compound promotes a modification in Bax:Bcl-2 ratio and increases p53 expression. Thus, it is possible to conclude that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate induces apoptosis by inhibiting the antiapoptotic protein Bcl-2 and by increasing the release of AIF, Bax and p53. In addition, it blocks the cell cycle at G0/G1, stopping cell proliferation. So far, the results suggest that this compound may have a potential therapeutic effect against leukemia cells.

RHAMNAZIN INHIBITS PROLIFERATION AND INDUCES APOPTOSIS OF HUMAN JURKAT LEUKEMIA CELLS IN VITRO.

Antiproliferative and apoptogenic effects of rhamnazin, a dimethoxylated derivative of quercetin, were studied in human acute lymphoblastic leukemia Jurkat cells. The cytotoxicity and apoptogenic activity of rhamnazin in vitro are inferior to that of quercetin. The apoptogenic activity of rhamnazin is realized via mitochondrial pathway and associated with activation of caspase-9 and -3. The additive apoptogenic effect of rhamnazin and suboptimal doses of etoposide, a DNA topoisomerase II inhibitor, is demonstrated. Therefore, methylation of quercetin modifies its biological effects considerably.

Success of an International Learning Health Care System in Hematopoietic Cell Transplantation: The American Society of Blood and Marrow Transplantation Clinical Case Forum

The American Society for Blood and Marrow Transplantation (ASBMT) Clinical Case Forum (CCF) was launched in 2014 as an online secure tool to enhance interaction and communication among hematopoietic cell transplantation (HCT) professionals worldwide through the discussion of challenging clinical care issues. After 14 months, we reviewed clinical and demographical data of cases posted in the CCF from January 29, 2014 to March 18, 2015. A total of 137 cases were posted during the study period. Ninety-two cases (67%) were allogeneic HCT, 29 (21%) were autologous HCT, and in 16 (12%), the type of transplantation (autologous versus allogeneic) was still under consideration. The diseases most frequently discussed included non-Hodgkin lymphoma (NHL; n = 30, 22%), acute myeloid leukemia (n = 23, 17%), and multiple myeloma (MM; n = 20, 15%). When compared with the US transplantation activity reported by the US Department of Health and Human Services, NHL and acute lymphoblastic leukemia cases were over-represented in the CCF, whereas MM was under-represented (P < .001). A total of 259 topics were addressed in the CCF with a median of 2 topics/case (range, 1 to 6). Particularly common topics included whether transplantation was indicated (n = 57, 41%), conditioning regimen choice (n = 44, 32%), and post-HCT complications after day 100 (n = 43, 31%). The ASBMT CCF is a successful tool for collaborative discussion of complex cases in the HCT community worldwide and may allow identification of areas of controversy or unmet need from clinical, educational and research perspectives.

Analysis of the Expression of PHTF1 and Related Genes in Acute Lymphoblastic Leukemia

Background: In the human T-cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4, siRNA-mediated suppression of BCL11B expression was shown to inhibit proliferation and induce apoptosis, which may be related to PHTF1 gene expression, and the FEM1B and Apaf-1 genes may be downstream of PHTF1. In this study, we analyzed the expression level of PHTF1 and related genes in patients with ALL to clarify the role of the PHTF1-FEM1b-Apaf-1 pathway in hematologic malignancies.Methods: Fifteen newly diagnosed and untreated patients with AML, fourteen newly diagnosed and untreated patients with CML in chronic phase, twenty-two newly diagnosed and untreated patients with ALL, and six newly diagnosed and untreated patients with CLL were recruited. Peripheral blood mononuclear cells (PBMCs) from ten healthy individuals (HIs) served as controls. PBMCs were separated using the Ficoll-Hypaque gradient centrifugation method. All procedures were conducted in accordance with the guidelines of the Medical Ethics committees of the Health Bureau of Guangdong Province, China.Real-time PCR was used to determine the gene expression level of PHTF1 in hematologic malignancies. The PHTF1, BCL11B, FEM1B and Apaf-1 gene expression levels and correlations were analyzed in patients with primary ALL and healthy individuals (HIs).Results: PHTF1 overexpression was found in recently diagnosed AML (p <0.001), CML-CP (p<0.001), and ALL (p= 0.016) patients in comparison with HIs. The PHTF1 expression level in CLL patients was not significantly different compared with HIs (p= 0.165). FEM1b and Apaf-1 overexpression was found in recently diagnosed ALL (p<0.005) patients compared with HIs. Positively correlated expression was found for the PHTF1, FEM1b and Apaf-1 genes in patients with ALL (p<0.005) and HIs (p<0.005), and positively correlated expression was found for the PHTF1 and BCL11B genes in HIs (p<0.005).Conclusions: PHTF1 acts as a tumor suppressor gene, and its overexpression might be related to cell proliferation, inhibition and apoptosis. PHTF1 and BCL11B gene disorders may contribute to T-ALL pathogenesis. PHTF1 might be a therapeutic target for triggering the PHTF1-FEM1b-Apaf-1 apoptosis pathway in primary acute lymphoblastic leukemia.AcknowledgmentThe project was sponsored by grants from National Natural Science Foundation of China (No. 81100384, No. 81270648, No.91129720, and No. 30771980) DisclosuresNo relevant conflicts of interest to declare.

Role of Mitochondrial Apoptotic Pathway in Disulfiram/Copper Mixture-Induced Cell Apoptosis in Human B-Lineage Acute Lymphoblastic Leukemia in Vitro

Backgrounds: The long term prognosis of adult B-lineage acute lymphoblastic leukemia (B-ALL) is poor when compared with pediatric B-ALL. The current therapeutic regimen for adult B-ALL often results in refractory and relapsing diseases. Therefore, it is urgently needed to explore novel approaches to treat adult B-ALL. Disulfiram (DS) has been used clinically as a safe anti-alcoholism drug for over 6 decades. Recent studies demonstrated that disulfiram/cooper mixture (DS/Cu) was cytotoxic to multiple solid cancers, but its effects on B-ALL cells are still unclear. Moreover, the molecular mechanism of the cytotoxicity of DS/Cu to tumor cells was poorly defined. In this study, we investigated the effects of DS/Cu on B-ALL cells in vitro and its related cytotoxic mechanism.Results: Firstly, CCK8 assay indicated that DS/Cu markedly inhibited Nalm6 cell proliferation in a dose-dependent manner. Secondly, colony-forming assay showed that DS/Cu also abolished the clonogenicity of Nalm6 cells (P<0.001). Thirdly, FACS analyses revealed that DS/Cu mixture could induce apoptosis of Nalm6 cells, as well as primary B-ALL cells (n=16) in a dose-dependent manner. We additionally analyzed the relationship between clinical characteristics of B-ALL patients, including age, WBC counts, immunophenotype, cytogenetics, risk stratification and Ph chromosome, with the efficacy of DS/Cu on B-ALL cells. The apoptosis isolated from pro-B and cytogenetic abnormality B-ALL pastients was higher. Therefore, our results demonstrated that DS/Cu mixture could induce significant cytotoxicity against B-ALL cells in vitro.To decipher the cytotoxic mechanism of DS/Cu mixture, JC-1 staining was done and the results showed that DS/Cu mixture could significantly reduce the mitochondrial membrane potential in Nalm6 cells (P<0.01) and 7 cases of primary B-ALL cells (P<0.05). Consistently, Western Blot analysis showed that DS/Cu induced B-ALL cell apoptosis by down-regulating the expression of anti-apoptotic protein Bcl2 and Bcl-XL, as well as activating caspase-3 and its substrate PARP. Hence, our results indicated that DS/Cu induced apoptosis of B-ALL cells at least partly through the intrinsic mitochondrial apoptotic pathway.Conclusion: Our results demonstrated that DS/Cu not only significantly inhibit proliferation and clonogenicity, but also induce apoptosis of B-ALL cells in vitro.The mitochondrial apoptotic pathway might be the molecular mechanism of DS/Cu-induced apoptosis of B-ALL cells. DisclosuresNo relevant conflicts of interest to declare.

Alarmin High Mobility Group Box 1 (HMGB1) Activates Alternative NFkB Signaling in Bone Marrow Macrophages in the Leukemia Microenvironment

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) that signals through pattern recognition receptors on monocyte/macrophages to recruit them to sites of tissue injury. In models of sepsis and autoimmunity, HMGB1 can have both immune-activating and regulatory effects depending on the receptor system engaged and the downstream pathway activated. Although chemotherapy-stressed tumor cells are known to release HMGB1, little is known about the effects of tumor-derived HMGB1 on macrophages in the tumor microenvironment.We have previously shown that primary human acute lymphoblastic leukemia cells release high levels of HMGB1 after treatment with anthracycline chemotherapy, and that leukemia-associated HMGB1 induces expression of the IL-1 converting enzyme (caspase-1), consistent with activation of the inflammasome. Paradoxically, inflammatory cytokine production (including IL-1beta) is reduced in this setting. In the present study, we test the hypothesis leukemia-associated HMGB1 activates alternative NFkB signaling through the repressor protein RelB which has been recently shown to silence inflammatory cytokine production via epigenetic effects in sepsis models.To establish proof-of-principle, we first treated primary human bone marrow macrophages (BMM) and THP1 monocytes with 200 ng/ml recombinant HMGB1 (r-HMGB1) in the presence or absence of bacterial lipopolysaccharide (LPS). As predicted, LPS alone increased baseline expression of total NFkB, total p65/RelA and phosphorylated p65/RelA mRNA (20.6 + 7.9-fold by qPCR ddCT, p<0.0001) and protein (measured by Western blot, in BMM and THP1. In contrast, r-HMGB1 treatment produced a significant increase in the expression of RelB mRNA (2.5 + 0.7 fold increase p<0.05) and protein without a significant change in the expression of tNFkB or p65/RelA. Treatment with both r-HMGB1 and LPS maintained this pattern of increased RelB expression without increased expression of p65/RelA, suggesting HMGB1 activity as the dominant effect. Increased RelB expression was unaffected by the addition of an antibody to TLR4, the primary receptor for LPS and a candidate receptor for HMGB1.Next, we determined whether induction of RelB expression also occurs in response to leukemia-derived HMGB1. Immunomagnetically selected ALL cells from the bone marrow of 3 pediatric patients and NALM 6 ALL cells were treated with 20 nM doxorubicin, washed, and placed in culture for 24 hours prior to the addition of BMM in direct coculture. In our model, ALL cells typically release 100-450 ng/ml HMGB1 under these conditions. Again, significantly increased expression of RelB mRNA (6.7 + 2.9 fold, p<0.001) and protein was observed in BMM cultured with doxorubicin-treated ALL cells compared to ALL cells treated with media control, with a corresponding decrease in phospho-IkBa protein expression and unchanged total NFkB, p65/RelA and phosphorylated p65/RelA. Pre-treatment of ALL cells with quercetin, a commercially available flavonoid antioxidant shown to inhibit HMGB1 release, inhibited protein expression of RelB in BMM in a dose-dependent fashion.These data indicate that tumor-derived HMGB1 activates alternative NFkB signaling and is capable of reprogramming the inflammatory response to LPS. Studies are ongoing to the determine downstream effects on macrophage antitumor function to optimize therapeutic reversal of HMGB1 effects and improve the potential for bone marrow macrophages to eradicate residual leukemia that remains after chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract A146: Study of anti-leukemia activity of N-(1-pyrenlyl) maleimide in vivo using a bioluminescent mouse model

Abstract Telomerase plays a pivotal role in cellular immortality and tumorigenesis. Telomerase activity is normally not detectable in most somatic cells, but is activated in the vast majority of cancer cells. Therefore, inhibition of telomerase has been viewed as a promising anticancer approach due to its specificity for cancer cells. During a search for anticancer drugs by screening for small molecules of telomerase inhibitors, we have identified a very promising compound, N-(1-pyrenyl) maleimide, which displays the greatest differential cytotoxicity against hematopoietic Jurkat-T cells in vitro. In this study, we examined the anticancer activity of N-(1-pyrenyl) maleimide against human T-cell acute lymphoblastic leukemia (T-ALL) in vivo. Human T- ALL cells, such as Jurkat cells, were transfected with luciferase-expressing plasmid to establish luciferase-expressing T-ALL cells. The luciferase-expressing T-ALL cells were mixed with matrigel and injected subcutaneously into the nude mice. We have successfully employed such a xenograft method to establish a bioluminescent mouse model for investigating the anti-leukemia activity of N-(1-pyrenyl) maleimide and other classical anti-leukemia drugs. Using the established bioluminescent mouse model, we have begun to examine the anticancer activity of N-(1-pyrenyl) maleimide against T-ALL. We will present our most updated findings in this meeting. (Supported by grant CMRPD3D0171 from Chang Gung Memorial Hospital) Citation Format: Tzu-Chien Wang, Pei-Chi Chang, Chi-Yuan Chen. Study of anti-leukemia activity of N-(1-pyrenlyl) maleimide in vivo using a bioluminescent mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A146.

NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity

AbstractIn developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.

Antileukemic Efficacy of Continuous vs Discontinuous Dexamethasone in Murine Models of Acute Lymphoblastic Leukemia.

Osteonecrosis is one of the most common, serious, toxicities resulting from the treatment of acute lymphoblastic leukemia. In recent years, pediatric acute lymphoblastic leukemia clinical trials have used discontinuous rather than continuous dosing of dexamethasone in an effort to reduce the incidence of osteonecrosis. However, it is not known whether discontinuous dosing would compromise antileukemic efficacy of glucocorticoids. Therefore, we tested the efficacy of discontinuous dexamethasone against continuous dexamethasone in murine models bearing human acute lymphoblastic leukemia xenografts (n = 8 patient samples) or murine BCR-ABL+ acute lymphoblastic leukemia. Plasma dexamethasone concentrations (7.9 to 212 nM) were similar to those achieved in children with acute lymphoblastic leukemia using conventional dosages. The median leukemia-free survival ranged from 16 to 59 days; dexamethasone prolonged survival from a median of 4 to 129 days in all seven dexamethasone-sensitive acute lymphoblastic leukemias. In the majority of cases (7 of 8 xenografts and the murine BCR-ABL model) we demonstrated equal efficacy of the two dexamethasone dosing regimens; whereas for one acute lymphoblastic leukemia sample, the discontinuous regimen yielded inferior antileukemic efficacy (log-rank p = 0.002). Our results support the clinical practice of using discontinuous rather than continuous dexamethasone dosing in patients with acute lymphoblastic leukemia.

Abstract 2095: DEPTOR is a direct NOTCH1 target that regulates proliferation, metabolism and glucocorticoid resistance in T cell leukemia

Abstract DEP-Domain Containing mTOR-Interacting Protein (DEPTOR) is frequently overexpressed in multiple myelomas and required for cell survival. However, its implications in other hematological malignancies and the precise mechanisms of regulation remain largely undefined. Here, we systematically analyzed DEPTOR expression in human acute lymphoblastic leukemia (ALL), a hematological malignancy accounting for 80% of pediatric leukemia. Whereas substantially inhibited in B-ALLs, unexpectedly, expression of DEPTOR is significantly elevated in a subset of T-ALLs where NOTCH1 is aberrantly activated. Mechanistically, we show DEPTOR expression is dependent on NOTCH signaling in T-ALL cell lines and primary specimens, and demonstrate NOTCH1 directly binds to and activates human DEPTOR promoter. DEPTOR depletion in T-ALL cells abolishes proliferation, attenuates glucose metabolism, enhances apoptosis and sensitizes resistant cells to γ-secretase inhibitor or dexamethasone treatments. Conversely, ectopically expressed DEPTOR promotes proliferation and aerobic glycolysis, and confers dexamethasone resistance. DEPTOR functions through AKT-dependent mechanisms to sustain cell proliferation and drug resistance, while the AKT-independent roles of DEPTOR appear to promote glucose metabolism. We therefore define a novel mechanism that, in addition to the previously reported HES1/PTEN-mediated regulation of AKT, NOTCH1 alternatively activates AKT through inducing the downstream target gene DEPTOR. Our findings thereby highlight DEPTOR as a critical mediator in leukemogenesis and suggest DEPTOR expression may serve as a potential biomarker predicting glucocorticoid resistance and targeting DEPTOR may achieve better therapeutic benefits. Citation Format: Yufeng Hu, Hexiu Su, Qi Ye, Zhaojing Wang, Liang Huang, Qian Wang, Shuangyou Liu, Suning Chen, Jianfeng Zhou, Peng Li, Guoliang Qing, Hudan Liu. DEPTOR is a direct NOTCH1 target that regulates proliferation, metabolism and glucocorticoid resistance in T cell leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2095. doi:10.1158/1538-7445.AM2015-2095

Abstract 3938: MafB and ETS2 potentiate Notch signaling in T-ALL

Abstract Despite the high frequency of activating Notch mutations in human T-cell acute lymphoblastic leukemia (T-ALL), these naturally occurring Notch1 mutant alleles fail to efficiently drive leukemia onset, indicating a requirement for collaborating events that potentiate the Notch oncogenic program. We performed an in vitro gain-of-function screen to identify gene products that enhanced the signaling strength of naturally occurring Notch mutants. Both MafB and ETS2 emerged as candidates that individually potentiated Notch signaling, and when co-expressed, they dramatically enhanced signaling strength comparable to the levels induced by MAML1, a core component of the Notch transcriptional complex. Knockdown of MafB in mouse T-ALL cell lines suppressed the expression of common Notch target genes, including cMyc and Hes1, and sustained knockdown of MafB expression impaired T-ALL cell growth in cell competition assays. Notably, MafB is sufficient to enhance the oncogenic potential of naturally occurring Notch mutants in vivo, by significantly accelerating T-ALL onset. Moreover, this enhanced Notch pathway activation required MafB-ETS2 interaction, and facilitated the recruitment of PCAF and p300. Although all primary human T-ALL samples that express MafB display an elevated Notch pathway signature, the T-ALL samples positive for high levels of MafB expression, exhibit low levels of Notch1 expression, further suggesting a role for MafB as an enhancer. These data present a novel mechanism for the enhancement of the oncogenic potential of weak Notch mutants in leukemia and introduce MafB as a potential therapeutic target in T-ALL. Note: This abstract was not presented at the meeting. Citation Format: Kostandin Pajcini, Lanwei Xu, Curtis Lee, Sara Cherry, Warren Pear. MafB and ETS2 potentiate Notch signaling in T-ALL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3938. doi:10.1158/1538-7445.AM2015-3938

Abstract 2309: Murine model of dual mTORC kinase inhibition identifies CDK6 as a synergistic target in T-ALL

Abstract The PI3K/AKT/mTOR pathway is frequently hyperactivated in T- cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with constitutively-active AKT specific to T-lymphocytes (Lck-MyrAkt2) were crossed with mice that have genetically-reduced mTOR expression (mTOR knock-down, KD). Lck-MyrAkt2 mice with mTOR KD had altered thymic T-lymphocyte development; these mice also had delayed thymic pre-T cell lymphoblastic leukemia/lymphoma (pre-T LBL) progression and increased survival relative to wild type (WT) mTOR/ Lck-MyrAkt2 mice (average survival of 24 versus 14 weeks, respectively). Delayed pre-T LBL formation was also observed when WT mTOR/Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex. Transcriptional profiling of thymic lymphomas from the WT vs KD mTOR/ Lck-MyrAkt2 mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T- cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. Tumor cells from WT mTOR/Lck-MyrAkt2 mice were more sensitive to CDK4/6 inhibitors than tumor cells from KD mTOR/Lck-MyrAkt2 mice. Combining an mTOR inhibitor (rapamycin) with a CDK4/6 inhibitor (PD-0332991) synergistically inhibited the viability and decreased downstream signaling in both mouse lymphoma cells and human T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Combining a dual mTORC inhibitor (PP242) with a CDK4/6 inhibitor decreased downstream signaling to an even greater extent in human T-ALL cell lines. Our results suggest that an mTORi/CDKi combination may be beneficial in the treatment of human T-ALL. Citation Format: Joy M. Gary, Jinfei Xu, John Simmons, Shuling Zhang, Benjamin Gamache, Ke Zhang, Alexander Kovalchuk, Aleksandra Michalowski, Jin-Qiu Chen, Michelle Herrmann, Wendy Dubois, Joseph Testa, Beverly A. Mock. Murine model of dual mTORC kinase inhibition identifies CDK6 as a synergistic target in T-ALL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2309. doi:10.1158/1538-7445.AM2015-2309

Spectral study of modified natural clinoptilolite with pharmacologically active escin and horse chestnut extract

ABSTRACTThe aim of this study was preparation of modified forms of natural zeolite of the clinoptilolite type with different content of pharmacologically active natural substances escin and horse chestnut extract and their characterization by infrared spectroscopy, fluorescence spectroscopy, thermal analysis, röntgen diffraction powder technique, and determination of the surface areas and the pore volumes by low-temperature adsorption of nitrogen. Escin and horse chestnut extract have also been investigated by atomic force microscopy. The analyses confirmed the presence of escin and horse chestnut extract in the modified zeolitic products. The modified forms of natural zeolite of the clinoptilolite type with low content of escin and horse chestnut extract have been used for preliminary in vitro study of their anticancer effects on cancer cell lines by an in vitro colorimetric assay for assessing cell viability. To understand the growth inhibitory effects, the human cervical carcinoma cells, human T-cell acute lymphoblastic leukemia, breast cancer cells, human alveolar basal epithelial cells, and human breast adenocarcinoma cells were treated with various doses of the modified zeolitic products. The modified forms were the most effective in adenocarcinomic human alveolar basal epithelial cells.

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol.

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728.

Doxorubicin induces apoptosis in Jurkat cells by mitochondria-dependent and mitochondria-independent mechanisms under normoxic and hypoxic conditions.

In this study, we investigated the molecular mechanism of doxorubicin (dxr)-induced cytotoxicity on Jurkat cells - a model cell of human acute lymphoblastic leukemia - under normoxic (20% O2) and hypoxic (5% O2) conditions. Using in-cell western analysis, immunofluorescence, flow cytometry analysis, and biochemical inhibitors, we evaluated several oxidative stress (OS) and cell death markers. It was found that dxr (5-100 μmol/l) induced apoptosis by OS mechanisms involving DNA fragmentation (8-48%), loss of mitochondrial membrane potential (ΔΨm, 33-92%), and H2O2 production (15-42%) under normoxia. In addition, dxr (10 μmol/l) induced activation and/or nuclei translocation of NF-κB (6.6, 1.6-fold increase), p53 (4.3, 3.1 f), c-Jun (9.5, 5.0 f), apoptosis-inducing factor (AIF) (1.9, 3.9 f), caspase-3 (3.7, 1.9 f), overexpression of Parkin (2.1, 1.2 f)/PINK-1 (2.1 f) proteins, and reduced DJ-1 levels by half compared with untreated cells under normoxia, according to immunofluorescence and in-cell western analysis, respectively. In contrast, dxr (10 μmol/l) could not induce apoptosis in Jurkat cells under hypoxia. Effectively, dxr significantly reduced DNA fragmentation (6%), expression levels of cell death (e.g. p53, c-Jun, caspase-3, AIF), and OS (e.g. Parkin) markers, whereas it increased ΔΨm, hypoxia-inducible factor 1-α (HIF-1α, 3.1, 2.3 f), NF-κB (6.8, 2.0 f), and DJ-1 (1.3, 1.0 f) levels. This investigation suggests that dxr might efficiently eliminate acute lymphoblastic leukemia cells by OS-induced apoptosis under normoxic conditions through a minimal completeness of cell death signaling (i.e. mitochondria-caspase-3/AIF-dependent pathways) and through a direct DNA damage process. However, hypoxic conditions may reduce the effectiveness of dxr toxicity.

Effects of tetramethylpyrazine injection on the proliferation and apoptosis of Jurkat cells and the relevant molecular mechanisms

Objective To study the effect of tetramethylpyrazine injection on proliferation and apoptosis of human acute lymphoblastic leukemia (ALL) cell line Jurkat and the relevant molecular mechanisms. Methods Cells were treated with tetramethylpyrazine injection at various concentrations (0, 0.25, 0.50, 0.75, 1.00, 1.25 and 1.50 mg/ml), CCK-8 method was used to detect the inhibition rates at 24, 48 and 72 h. After cells were treated with different concentrations of tetramethylpyrazine injection (0, 0.50, 1.00 and 1.50 mg/ml) for 48 h, the cell cycle and apoptosis were detected by flow cytometry, and the Aurora-B and Survivin protein expression were analyzed by Western blot. Results Compared with the control group (cells without tetramethylpyrazine injection treatment), various concentrations of tetramethylpyrazine injection could effectively inhibit Jurkat cells proliferation in a time-and dose-dependent manner (P < 0.05), and IC50 at 24 h,48 h and 72 h after treatment were (1.33±0.16), (0.91±0.10) and (0.67±0.11) mg/ml, respectively. After cells were treated with tetramethylpyrazine injection at different concentrations (0.5, 1.0 and 1.5 mg/ml) for 48 h, the number of treated cells in G2/M phase was increased, but that in S phase was decreased, and the apoptosis rates were significantly higher than that of control group, with a dose dependence (P < 0.05). The results of Western blot showed that the expression levels of Aurora-B and Survivin in treated cells were lower than that of control group, also with a dose dependence (P < 0.05). Conclusions Tetramethylpyrazine injection can effectively inhibit proliferation and induce apoptosis of Jurkat cells in vitro, and its underlying mechanisms involve with down-regulation of the Aurora-B and Survivin protein expression. Key words: Tetramethylpyrazine injection; Leukemia; Cell proliferation; Cell apoptosis; Aurora-B; Survivin

Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

Preclinical activity of SL-801, a reversible inhibitor of Exportin-1 (XPO1)/Chromosome Region Maintenance-1 (CRM1) in solid and hematologic cancers.

e13543 Background: XPO1 controls key cellular processes by regulating nuclear export of proteins and RNA. XPO1 is overexpressed in many cancers, causing dysregulated protein localization, aberrant growth, and resistance to apoptosis. Recent work has revealed XPO1 to be a clinically relevant target, and nuclear export inhibitors have emerged as a new class of anti-cancer agents with clinical activity in multiple indications. SL-801 is a structurally novel small molecule that binds covalently but reversibly to XPO1, blocking binding of substrate cargos. The kinetics of target binding and preclinical activity of SL-801 in multiple tumor types are reported here. Methods: SL-801 activity was evaluated against 240 tumor cell lines using the OncoPanel platform and in human tumor xenografts in SCID mice. Pull-down assays with biotinylated SL-801 were used to assess SL-801 binding to XPO1. Results: SL-801 demonstrated broad and potent activity across numerous tumor cell lines. GI50s in hematologic cancers ranged from 3-93 nM in leukemias, 1-103 nM in lymphomas, and 3-11 nM in multiple myelomas. SL-801 also inhibited solid tumor growth, with GI50s ≤ 10 nM in breast, brain, cervical, ovarian, gastric, kidney, liver, lung, melanoma, prostate, and sarcoma lines. SL-801 exhibited substantial activity in vivo. In an ARH-77 multiple myeloma xenograft, 90% of mice treated with SL-801 in a multi-dose regimen survived 150 days, while the median survival of vehicle-treated mice was 40 days. SL-801 was also active in human acute lymphoblastic leukemia, non-small cell lung cancer, and prostate carcinoma xenograft models. Pull-down assays showed that SL-801 binding to XPO1 decreased after 2 hours, whereas the prototypical XPO1 inhibitor leptomycin B bound tightly to XPO1 for at least 8 hours. Conclusions: SL-801 is a promising clinical candidate that inhibits a novel target and possesses potent and broad anti-cancer activity. The reversible binding of SL-801 to XPO1 may offer the potential to develop dosing schedules to enable recovery in normal tissues, broadening the therapeutic index of this class of agents. IND-enabling work is underway to support entry into clinical trials.