Treatment Of T-ALL Research Articles

Evaluation of KPT-SINE (Selective Inhibitors of CRM1-Mediated Nuclear Export) in AML and T-ALL.

e13586 Background: CRM1 is the major nuclear exporter that mediates transport of a variety of molecules, including proteins involved in tumor suppressor and cellular proliferation pathways. The crystal structure of CRM1, in complex with its export cargo, Snurportin 1, has been recently resolved. The molecular understanding of the CRM1-cargo binding interface has led to the development of novel small molecule inhibitors of CRM1-cargo interaction, termed KPT-Selective Inhibitors of Nuclear Export (SINE). KPT-SINE are potent, drug-like CRM1 inhibitors that irreversibly inactivate the CRM1-directed protein export by covalent modification of the essential CRM1-cargo binding residue Cys528. The inhibition of the CRM1 nuclear export has been shown to lead to selective apoptosis in cancer cells when compared to normal cells. Here, we assess the efficacy of the KPT-SINE in human AML, T-ALL, and normal hematopoietic cells. Methods: The viability of a panel of human AML and T-ALL cell lines upon treatment by the KPT-SINE was assessed. Dose-response measurements were done using serial dilutions of KPT-SINE from 1 µM to 0.3 nM and luminescent cell viability assay. Apoptosis was measured using Annexin V staining and TUNEL assays. Results: KPT-SINE induces rapid apoptosis in 10 of 14 AML and 12 of 15 T-ALL cell lines with IC50s of 15-100 nM. In the KPT-SINE-sensitive cell lines, BCL2 overexpression suppresses KPT-SINE-induced apoptosis, indicating its intrinsic pathway mediation. Importantly, KPT-SINE compounds, either KPT-251 at 75 mg/kg or KPT-330 at 15 or 25 mg/kg, induced remarkable growth suppression in MV4-11 human AML cells engrafted into immunodeficient NSG mice with minimal toxicity to normal mouse hematopoietic cells after 35 days of treatment. Bone marrow biopsies of KPT-treated mice were remarkable in that they showed normal hematopoietic cell morphology and cellularity after 35 days of treatment. Xenograft studies to address the potency of the KPT-251 and KPT-330 on MOLT-4 human T-ALL cells in vivo are ongoing. Conclusions: These studies emphasize the clinical promise of the KPT-SINE as a novel and selective drug candidate for the treatment of AML and T-ALL.

Nelarabine Is Safe and Effective In Adult Relapsed or Refractory T Cell Acute Lymphoblastic Leukemia (T-ALL) and Lymphoblastic Lymphoma (T-LBL): The Bologna Experience

Abstract 4250 Background.Nelarabine (N) is approved for the treatment of T-ALL and T-LBL that have not responded to or has relapsed after treatment with at least 2 chemotherapy regimens.Aim. To evaluate safety profile and efficacy of N as savage therapy in 16 adult relapsed or refractory T-ALL or T-LBL. Methods.After obtaining an informed consent, 16 patients (median age 33 years, range 19–45, M/F= 13/3) affected by T-ALL (n=10) and T-LBL (n=6) received savage therapy with N (median cycle=1, range 1–3), administered at standard adult dosage (1500 mg/sqm on days 1, 3 and 5, every 21). Four patients were primary resistant to induction treatment, 7 patients were relapsed after two previous chemotherapy regimens (including allogeneic BMT in 4 cases and autologous SCT in 1 case); the remaining 6 patients had a molecular relapsed disease (MRD positive). Molecular characterization was performed, including NOTCH and WT-1 genes mutational status. GEP analysis, according to Ferrando A. stratification (Cancer Cell 2002), is still ongoing. Results.Currently, 12 out of 16 patients are evaluable, due to a too short follow up in the other 4 cases. Seven out of 12 patients obtained a complete remission (CR) (5 T-ALL 2 T-LBL);a partial remission (PR) was documented in 2 cases, with an overall response rate (ORR) of 75%. Median duration of CR was 10 weeks (range 2.8–54+). Among these, 2 out of 4 patients in molecular relapse reached a molecular CR and underwent an allogenic BMT (currently in CR after a median follow up of 12 months). Extra- hematological toxicity, not clearly related to the drug, occurred in 3 cases, determining, a complete and irreversible paraplegia, a condition of mental confusion, and a peripheral neuropathy, respectively. Conclusions.N showed a strong efficacy also in cases with low levels of residual disease, in addition to a good safety profile. Neurological toxicity needs to be strictly monitored. Acknowledgments.European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, PRIN 2009, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures:Baccarani:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

KPT-SINE, a Potent, Small Molecule Inhibitor of CRM1-Dependent Nuclear-Cytoplasmic Shuttling, with Potent Activity Against T-ALL and AML

Abstract 2622 Background:CRM1 (XPO1) is the major nuclear exporter that mediates transport of a variety of cargos, including proteins involved in tumor suppressor and cellular proliferation pathways (e.g. p53, PI3K/AKT, and NF-kB). CRM1 is upregulated in a range of solid and hematologic malignancies and its overexpression is correlated with poor prognosis, and with resistance to chemotherapy. Recently, the crystal structure of CRM1, in complex with its export cargo, Snurportin 1, has been resolved. The structure elucidates the key contact residues required for the formation of the CRM1-cargo export complex. The molecular understanding of the CRM1-cargo binding interface has led to the development of novel small molecule inhibitors of CRM1-cargo interaction, termed KPT-Selective Inhibitors of Nuclear Export (SINE). KPT-SINE are potent, drug-like CRM1 inhibitors that irreversibly inactivate the CRM1-directed protein export by covalent modification of the essential CRM1-cargo binding residue Cys528. The inhibition of the CRM1 nuclear export has been shown to lead to selective apoptosis in cancer cells when compared to normal cells. Here, we assess the efficacy of the KPT-SINE in human T-ALL, AML, and normal hematopoietic cells. Methods: The viability of a panel of human T-ALL and AML cell lines and normal CD34+ progenitor cells upon treatment by the KPT-SINE was assessed. Dose-response measurements were done using serial dilutions of KPT-SINE from 1 μM to 0.3 nM and luminescent cell viability assay. Apoptosis was measured using Annexin V staining and TUNEL assays. Results: KPT-SINE induces rapid apoptosis in 12 of 15 T-ALL and 10 of 14 AML cell lines with 50% inhibitory concentrations (IC50s) of 15–100 nM. In the KPT-SINE-sensitive cell lines, BCL2 overexpression suppresses KPT-SINE-induced apoptosis, indicating its intrinsic pathway mediation. Importantly, KPT-SINE exhibits selective cytotoxicity against tumor cells when compared to peripheral blood mononuclear cells (PBMCs) and CD34+ progenitor cells in vitro. KPT-SINE at 57 and 150 mg/kg induced 100% inhibition of tumor growth in vivo in a MOLT-4 T-ALL xenograft study. Other xenograft studies to address the potency of the KPT-SINE on selected AML lines in vivo are ongoing. These studies emphasize the clinical promise of the KPT-SINE as a novel and selective drug candidate for the treatment of T-ALL and AML. Disclosures:McCauley:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm: Equity Ownership. Shacham:Karyopharm: Equity Ownership.

Cooperating Oncogenes and Their Targets in LMO2-Induced T-Cell Leukemia

Abstract 2458LIM domain Only 2 (LMO2) is an important driver in acute T-cell lymphoblastic leukemia. Enforced expression of LMO2 in mouse models induces T-ALL with long latency suggesting that it requires cooperating oncogenic mutations for leukemia to fully manifest. To identify cooperating oncogenes, we analyzed spontaneous T-ALLs induced by retroviral insertional mutation, and T-ALLs that developed in B6.CD2-Lmo2 transgenic mice. First, we noted that the CD2-Lmo2 transgenic T-ALLs exclusively upregulated the class II basic helix-loop-helix transcription factor, Lyl1, whereas the retrovirally-induced T-ALLs showed upregulation of Tal1. We attributed this difference to the stage at which the Lmo2 activating mutations occurred. Nevertheless, we found that LYL1 protein upregulation is functionally important, as it is part of a macromolecular complex containing LMO2 that binds tandem E boxes as described for TAL1. LMO2 and LYL1 are known to be co-expressed in human T-ALL, but we found that they are part of a larger gene expression signature comprised of NMYC, HHEX, MEF2C, and BCL2 genes. The latter genes are remarkable because they are oncogenic when overexpressed; and, Mef2c and Nmyc are concordant insertions in independent tumors driven by Lmo2 or Hhex in retroviral insertional mutagenesis models. In fact, insertion site analysis suggests that Lmo2 and Hhex may actually operate in the same pathway and that Hhex may be a target of Lmo2. We tested this genetic result by chromatin immunoprecipitation and reporter assays and discovered that Lmo2 and its binding partners, Ldb1 and Lyl1, occupy an enhancer in the first intron of Hhex in T-ALL cells. Furthermore, LMO2 or LYL1 knockdown downregulated expression of HHEX in T-ALL. HHEX activation has significant functional consequences in T-ALL. We found that its upregulation is associated with T-ALL treatment resistance, clinical latency, and differential activation of Notch1 targets. These data offer important insight into the pathogenesis of Lmo2-induced T-ALL. Disclosures:No relevant conflicts of interest to declare.

Growth Factor Independent-1 (Gfi1) As a New Target for Human Leukemia Therapy

Abstract 560More than 50% of patients diagnosed with B or T-cell leukemia and lymphoma will fail current treatment protocols. This highlights the urgent need for new and improved therapies. Since the transcription factor Growth factor independent-1 (Gfi1) plays an important role in lymphoid differentiation, we explored whether it might be a suitable target for therapy. Using mouse models in which T-cell leukemia can be induced by transgenic expression of mutated forms of Notch1, by injection of the carcinogen N-Ethyl–nitrosourea (ENU) or infection with a Murine Moloney Leukemia Virus, we found that Gfi1 knockout mice had a significantly lower incidence and a longer latency period of T-ALL. To verify whether targeting Gfi1 would be a novel approach to treat B- or T-cell lymphoma, we used Mx1Cre Gfi1fl/fl mice. In these mice, injection of polyinosinic-polycytidylic acid (pIpC) activates the Mx1 promoter driven Cre expression, which ultimately leads to the deletion of the floxed Gfi1 alleles. As controls, we used Gfi1fl/fl mice, which lack the Cre recombinase and thus still express Gfi1 after pIpC injection. To elicit a T- or B-cell lymphoma, we used ENU injection combined with expression of a mutated Notch1 transgene for T-ALL, or transgenic over-expression of c-Myc for B-cell leukemia (Eμ-Myc). Using in-vivo ultrasound supported imaging, we observed complete regression of tumour masses when Gfi1 was eliminated in Mx1Cre Gfi1fl/fl mice, curing the mice of the either the T or B-cell malignancies. Strikingly, this effect was observed in the absence of any other treatment regimen.To explore the mechanisms underlying this phenomenon, we explanted tumor samples from mice, in which Gfi1 expression was either maintained or deleted and performed gene expression arrays. A comparative analysis of the array data demonstrated that loss of Gfi1 affects pathways of key importance for leukemia such as metabolism, cell cycle progression, basal transcription and apoptosis but also the response to DNA damage. It has been shown previously for non hematological malignancies that oncogenic transformation in conjuction with dysregulated cell cycle induces DNA strand breaks (DSBs), which leads to an increased p53 dependent apoptotic response in tumours compared to non-transformed cells. This forces the tumor cells to counteract this effect – for instance by selection for the loss of p53. Consistent with this concept, we noted that leukemic cells from our tumor models displayed a greater amount of DSBs and also higher rates of spontaneous apoptosis than normal cells. Interestingly, the number of apoptotic cells was further increased in those tumors where Gfi1 had been deleted. We hypothesized that Gfi1 protects leukemia cells against DSB induced apoptosis. To test this hypothesis, we irradiated in Gfi1+/+ and Gfi1−/− thymocytes, which induces DSB in thymocytes. In line with our hypothesis we found that Gfi1−/− thymocytes showed increased rates of apoptosis compared to irradiated Gfi1+/+ thymocytes and that loss of Gfi1 led to an increased induction of pro-apoptotic genes such as Bax, Noxa and Puma after irradiation. Since Bax, Noxa and Puma are all p53 target genes, we investigated a possible link between Gfi1 and p53 and found that (I) Gfi1 binds to p53, (II) that Gfi1 inhibits the transcription of p53 target genes and (III) that Gfi1 occupies p53 target gene at the same sites as p53. In summary, Gfi1 antagonizes p53 function and loss of Gfi1 sensitizes cells to p53-mediated apoptosis.Next, we used different human T-ALL cell lines and treated these cells either with sh-RNA lentivirus or morpholinos to abrogate GFI1 expression. In all cases, down-regulation of GFI1 expression led to increased apoptosis and impeded growth of human leukemia cells. Finally, we transplanted leukemic cells of T-ALL patients into NOD-Scid, IL2Rnull (NSG) mice, waited for the leukemic cells to engraft, and then injected GFI1 specific- or control morpholinos. While mice treated with control morpholino died of leukemia, the animals treated with GFI1-specific morpholinos survived showing a significant reduction of human leukemic cells in the blood, bone marrow and spleen, even with samples from patients who did not respond to first line therapy. Since morpholinos have received approval for use in humans, our data suggest that targeting GFI1 in human T-ALL patients may be a promising therapeutic target and a feasible way to complement current T-ALL treatment regimens. Disclosures:No relevant conflicts of interest to declare.

Critical roles of NOTCH1 in acute T-cell lymphoblastic leukemia

NOTCH1 plays a central role in T-cell development and, when aberrantly activated, in acute T-cell lymphoblastic leukemia (T-ALL). As a transmembrane receptor that is ultimately converted into a transcription factor, NOTCH1 directly activates a spectrum of target genes, which function to mediate NOTCH1 signaling in normal or transformed T cells. During physiologic T-cell development, NOTCH1 has important functions in cell fate determination, proliferation, survival and metabolism. Activating NOTCH1 mutations occur in more than half of human patients with T-ALL, suggesting an important role for aberrant NOTCH1 signaling in the pathogenesis of this disease. Inhibiting NOTCH1 signaling in patient-derived cell lines and murine T-ALLs leads to growth arrest and/or apoptosis suggesting that NOTCH1 inhibitors can improve T-ALL treatment. However, there are challenges to translate NOTCH1 inhibitors to the clinic because of toxicity and resistance. This review focuses on molecular mechanisms of oncogenic NOTCH1 signaling, molecular and functional analysis of NOTCH1 transcriptional targets in T-ALL, and recent advances in therapeutic targeting of NOTCH1.

A Drug Screen in Zebrafish Identifies a New Therapeutic Agent Active in Acute Lymphoblastic Leukemia

AbstractAbstract 2129 Objectives:T cell lineage is an independent high risk factor in acute lymphoblastic leukemia (ALL). T-ALL requires high dose multi-agent chemotherapy, conferring many toxic side effects. T-ALL treatment therefore needs new, targeted agents that preserve or improve current treatment efficacy, yet cause fewer side effects than existing chemotherapeutic regimens. To identify such drugs, we pioneered an in vivo screen using transgenic zebrafish with T cell-specific green fluorescent protein (GFP) expression. We reasoned that immature T cells in 5-day-old zebrafish larvae are developmentally analogous to T-ALL lymphoblasts, and likely rely upon similar signaling pathways. Hence, compounds that specifically eliminate T cells in zebrafish larvae might likewise selectively target T-ALL cells. An added benefit of our in vivo screen is that drugs added to the water housing fish larvae must cross an epithelial barrier, can be metabolized by the liver, and can be renally cleared, characteristics not assessed in in vitro-based screening strategies. In addition, by using early larvae as our subjects, drugs lacking T cell specificity will likely impair normal development and/or survival, which we postulate is a predictor of unwanted toxicities. In these ways, our screen mimicked the scenario encountered in patients. Our use of the transgenic p56lck::EGFP zebrafish line facilitated rapid visual assessment of efficacy of a large number of compounds in 96-well format. Materials and Results:In a proof-of-principle experiment, we identified several known anti-T-ALL drugs, most prominently glucocorticoids, from the “Spectrum” library (MicroSource Discovery Systems, Inc., Gaylordsville, CT) of well-characterized compounds. We then screened 39,200 small molecules from the “ChemBridge DIVERSet” combinatorial chemistry library (ChemBridge Corp., San Diego, CA) for those active against zebrafish larval T cells. Of 20 novel “hits” identified, one compound, dubbed Lenaldekar (“LDK”), met additional prioritization criteria. LDK does not impair the cell cycle of developing zebrafish, is well tolerated and orally bioavailable with favorable pharmacokinetic properties in mice. In addition, pilot studies with LDK indicate it is efficacious in treating T-ALL in juvenile and adult fish from an established transgenic rag2::ER-human cMyc T-ALL model. LDK kills all of several murine T-lymphocytic malignancies, induces apoptosis in all human T-ALL lines tested, and shows some activity in human B-ALL lines. However, glioblastoma, colon carcinoma, melanoma, or immortalized human embryonic kidney cell lines are not affected by LDK, even at high concentration (up to 25μM). Using the recently established “phosflow” technique we measured phosphorylation status of key signaling proteins in permeabilized T- and B-ALL lines. Regardless of PTEN status, PDK1, AKT and mTOR downstream targets p4EBP1 and p70S6kinase were dephosphorylated by LDK treatment, as was the p65 subunit of NFκB on serine 529. Results were corroborated by conventional Western blots. However, phosphorylation of STAT3, STAT5, pERK1-2, and p38 were not affected by LDK. LDK's action is distinguished from other AKT/mTOR inhibitors by its lack of activity against AKT-dependent glioblastoma and melanoma cell lines, and its lack of effect on cell size. Finally, LDK decreased tumor burden of human T-ALL in murine xenografts. Conclusions:In view of its apparent lymphocyte selectivity, we posit that LDK modulates a pathway relatively unique to ALL (and immature lymphoblast) cells. This suggests that LDK can serve as a novel molecular tool for studies of normal and malignant lymphocyte biology. Moreover, with its favorable pharmacokinetics, apparent lack of toxicity, and in vivo efficacy in two vertebrate ALL models, LDK is an attractive molecule for development into a targeted treatment for ALL and perhaps other lymphocytic malignancies. Disclosures:No relevant conflicts of interest to declare.

Targeted Inhibition of mTORC1 and mTORC2 by the Active-Site mTOR Inhibitors, PP-242 and OSI-027, Has Cytotoxic Effects In T-Cell Acute Lymphoblastic Leukemia

AbstractAbstract 3242T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia characterized by uncontrolled proliferation of lymphoblasts committed to the T-cell lineage. Despite improvements in therapy, 25% of children and 50%–70% of adults with T-ALL relapse and develop a polychemotherapy-resistant disorder, which carries a poor prognosis. Thus, major efforts are being made to develop targeted molecules against deregulated signaling pathways that sustain T-ALL cell growth and survival. The mTOR serine/threonine kinase belongs to two separate multi-protein complexes, referred to as mTORC1 and mTORC2. The mTORC1 complex, which is inhibited by rapamycin/rapalogs, controls mRNA translation through the phosphorylation of the translation repressor 4E-BP1, while mTORC2 could be involved in regulating proliferation and survival through phosphorylation of Akt on Ser 473. mTORC2 is usually considered to be rapamycin-resistant. Both mTORC1 and mTORC2 are activated in most of T-ALL patients underscoring these complexes as major targets for T-ALL treatment. However, the allosteric inhibition of mTORC1 by rapamycin/rapalogs has only modest effects in T-ALL cells. This could be due, among other things, to the fact that rapamycin/rapalogs are mainly cytostatic and do not dephosphorylate 4E-BP1 in preclinical models of T-ALL. Recently, however, ATP-competitive inhibitors specific for the mTOR kinase active site have been synthesized. These compounds target both mTORC1 and mTORC2 and are more active than rapamycin/rapalogs against p-4E-BP1. Here, we have explored the therapeutic potential of two active-site mTOR inhibitors, PP-242 and OSI-027, against both T-ALL cell lines and primary samples from T-ALL patients displaying activation of mTORC1 and mTORC2. OSI-027 is being now tested in clinical trials in patients with solid malignancies. PP-242 and OSI-027 affected T-ALL cell line viability as documented by MTT assays. IC50 for PP-242 ranged from 0.15 to 0.50 μM at 24 hours, whereas OSI-027 was less potent (IC50 ranging from 0.6 to 1.3 μM at 48 h). Of note, both the drugs were effective against CEM T-ALL cells overexpressing 170-kDa P-glycoprotein, one of the major determinants of drug-resistance. Overall, PP-242 was slightly more powerful than the dual PI3K/mTOR inhibitor, PI-103, whose IC50 ranged from 0.2 to 1.0 μM. When T-ALL patient samples were examined, PP-242 displayed an IC50 of 0.5–1.0 μM, while the IC50 for OSI-027 ranged between 2.3 and 4.8 μM. Both the inhibitors affected to a much lower extent the proliferation of peripheral blood T-lymphocytes from healthy donors stimulated with phytohemagglutinin and interleukin-2. Remarkably, PP-242 targeted the side population of T-ALL cell lines, which might correspond to leukemic stem cells. The drugs induced both cell cycle arrest in G0/G1 phase and apoptosis, as documented by flow cytometric analysis, western blot analysis for cleaved caspase-3, and transmission electron microscopy. Western blot analysis demonstrated that the inhibitors induced a dose- and time-dependent dephosphorylation of Akt on Ser 473 (indicative of mTORC2 inhibition) as well as a dephosphorylation of mTORC1 downstream targets including Thr 389 p70S6 kinase, Ser 235/236 S6 ribosomal protein, Thr 37/46 4E-BP1, and Ser 209 eIF4E. Also a dephosphorylation of the Akt downstream target GSK3β on Ser 21/9 was observed. In contrast, rapamycin failed to dephosphorylate Thr 37/46 4E-BP1 and Ser 209 eIF4E. At variance with rapamycin, we also found a marked inhibition of mRNA translation in T-ALL cell lines treated with active-site mTOR inhibitors, as attested by a reduction in the incorporation of 3H-leucine and a shift from large to small polysomes.In MOLT-4 and Jurkat cells, the inhibitors, when used at subcytotoxic concentrations, strongly synergized with vincristine (combination index = 0.2–0.6), a traditional chemotherapeutic drug currently used for treating T-ALL patients, and with the Bcl-2 inhibitor, ABT-263 (combination index =0.15-0.19). In conclusion, both the inhibitors displayed a remarkable anti-leukemic activity through inhibition of mTORC1 and mTORC2 activity. The lower cytotoxicity against healthy T-lymphocytes ex-vivo suggests a favourable therapeutic index, which emphasizes the development of active-site mTOR inhibitors as clinical candidates for therapy in T-ALL. Disclosures:No relevant conflicts of interest to declare.

SHIP1 and PTEN Co-Inactivation and Akt Pathway Targeting in ALL.

Abstract 2386Poster Board II-363Despite success in the treatment of Acute lymphoblastic leukemia (ALL), relapse and death continue to occur and in survivors, secondary malignancies due to the impact of aggressive chemotherapy negatively impact the quality and duration of life. Akt plays a central role in signal transduction and is negatively regulated by PTEN and, in cells of hematopoietic lineage, SHIP1; it is positively regulated by PIK3CA. Despite multiple levels of regulation, Akt is aberrantly activated in many cancers including ALL. To understand the mechanism behind Akt activation in ALL, PIK3CA, PTEN and SHIP1 genes were assessed in leukemia cell lines and primary samples. No sample harbored PIK3CA mutation. PTEN was expressed in just one-third of the cell lines analyzed, but in two-thirds of the primary ALL of both T- and B-lineage, though all were in the phosphorylated (inactivated) form. SHIP1 was expressed in all leukemia cell lines, except for Jurkat and K562 cell lines. In general, expression of SHIP1 protein was much higher in cell lines of the B-lineage than of the T-lineage. The Jurkat T-ALL cell line, long observed to lack SHIP1 protein, harbored biallelic inactivation by null mutation and a frame-shift deletion; no SHIP1 mutations were identified in the K562 cell line. In contrast to leukemia cell lines, no full length SHIP1 was detected in primary T-ALL, and only ∼20% of the primary B-precursor ALL expressed full length SHIP despite the expression of full length transcript. Both ALL lineages expressed truncated isoforms of SHIP, which cloning and sequence analysis revealed was due to premature termination resulting from the frame-shift and other translationally-inactivating alternative splicing. The co-inactivation of SHIP and PTEN may create therapeutic opportunities. The PI3KCA inhibitor LY294002 has been shown to sensitize glioblastoma and colon carcinoma to doxorubicin. To determine if these agents acted synergistically in T-ALL, cell lines Molt16 (SHIP1+/PTEN-), Jurkat (SHIP1-/PTEN-) and normal MNCs (SHIP1+/PTEN+) were subjected to various combinations of the PIK3CA inhibitor LY294002 and doxorubicin and cell proliferation and viability assessed. Normal cells were generally insensitive to both agents except at high concentrations. LY294002 and doxorubicin inhibited viability in Molt16 with IC50s of 10 uM and 0.1 ug/ml, respectively, with no evidence of synergy. Jurkat cells exhibited a similar IC50 to LY294002, but were more sensitive to doxorubicin (IC50s of 10 uM and 0.025 ug/ml, respectively), with the two agents demonstrating some synergy when added simultaneously at low concentrations. In summary, we present evidence of Akt pathway deregulation in T-ALL due to SHIP1/PTEN inactivation and suggest that agents targeting this pathway may provide new directions into the therapeutic treatment of T-ALL. Disclosures:No relevant conflicts of interest to declare.

Interactions of slope and canopy of herbage of three herbage species on transport of faecal indicator bacteria by rain splash

AbstractThe movement of faecal pathogens from land to surface and ground‐water are of great interest because of the public and livestock health implications. Knowledge of canopy structure and how it might be managed to help mitigate nutrient and pathogen movement in pasture is needed to create management practices that balance livestock production with environmental benefits. An experiment was conducted using a rainfall‐simulating device to test whether canopy structure of species common to pastures in Appalachia, USA could be managed to influence dispersion of faecal pathogens. Seven pots (30‐cm diameter) of white clover, orchardgrass and perennial ryegrass were lined up on horizontal and sloping surfaces under a rainfall simulator. The centre pot was inoculated at the soil surface with 4 × 1010 faecal coliform bacteria (FC) just before rainfall simulation started. The species were maintained under short, moderate and tall canopy management treatments. White clover exhibited the greatest rates of lateral and vertical dispersion of FC into the canopy, especially in the short canopy management treatment following 30 min of rainfall (about 40 mm). Low concentrations of faecal coliform bacteria also dispersed into the canopies of the grass species but the differences in concentration of FC between the grass species were not different. When the proportion of white clover in a pasture is high, the canopy should be relatively taller to reduce the likelihood of infection associated with faecal coliform‐contaminated herbage.

CAL-101, a Potent Selective Inhibitor of the p110d Isoform of Phosphatidylinositol 3-Kinase, Attenuates PI3K Signaling and Inhibits Proliferation and Survival of Acute Lympoblastic Leukemia in Addition to a Range of Other Hematological Malignancies

The class I phosphatidylinositol 3-kinases (PI3K) regulate a variety of cellular functions including motility, metabolism, proliferation, growth, and survival, depending on cellular milieu. Deregulation of the PI3K/Akt pathway is one of the most frequently observed defects in human malignancies including those of hematological origin and has been shown to play an important role in tumor progression. Therefore, selective targeting of PI3K signaling in hematological tumor cells could provide an effective treatment strategy while limiting potential undesirable effects of pan-inhibitors that broadly block PI3K signaling in all cells. Of the class IA PI3Ks (p110a, p110b, p110d), p110d's expression is largely restricted to cells of hematopoietic origin and is essential for PI3K signaling in lymphocytes. Here, we report on the characterization of a novel p110d specific inhibitor, CAL-101. This compound is a potent PI3K inhibitor with an IC50 of 1–10 nM against the purified p110d subunit and 30–70 nM cellular potency against p110d-mediated basophil activation in whole blood. Importantly, CAL-101 plasma concentrations of 500–5000 nM that greatly exceed those needed for p110d inhibition in blood were safely maintained in a 7 day multidose normal human volunteer study. CAL-101 demonstrates >30-fold selectivity over other class I, II and III PI3K family members as well as selectivity over other PI3K-related proteins including mTOR and DNA-PK. Furthermore, a genome wide screen of >350 protein kinases did not detect any activity. To investigate the potential role of p110d in hematologic tumors we screened a wide range of leukemia and lymphoma cell lines for p110 isoform expression and constitutive PI3K pathway activation. The expression of p110d was observed in >90% of these cell lines that was in many cases accompanied by constitutive Akt phosphorylation. In this context, CAL-101 was able to reduce p-Akt levels and block additional downstream effectors such as p-p70S6K, p-GSKb, and p-Bad in cells that represent a range of tumor types including acute myeloid leukemia, acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma among others. Recent studies have demonstrated the importance of PTEN loss and enhanced PI3K signaling in primary T-ALL cells. We report high levels of p110d protein and activated Akt in 6 of 6 ALL cell lines evaluated. Inhibition of p110d with CAL-101 treatment of both T-ALL and B-ALL cell lines resulted in a reduction of Akt and GSK-3b phosphorylation and a decrease in cellular proliferation that was accompanied by cell death demonstrating an essential role of PI3K signaling independent of PTEN status. Treatment of T-ALL cell lines with CAL-101 induced processing of pro-caspase-3 and cleavage of PARP supporting a role for caspase mediated cell death. These studies have now been extended to the analysis of primary patient blast samples to further establish preclinical proof of concept for therapeutic application of CAL-101 for the treatment of ALL. In summary, CAL-101 is a highly potent and selective p110d kinase inhibiter with broad anti-tumor activity against cancer cells of hematologic origin. Clinical studies in normal human volunteers demonstrated good tolerability with high drug exposure and favorable steady-state pharmacokinetic properties. Taken together, these data support the on going Phase 1 clinical trial that includes a wide range of hematological malignancies.

NTAL Potentiates Glucocorticosteroid-Induced Apoptosis in T-ALL – in Vivo Observations and Experimental Model

PAG, LAT, NTAL and LIME belong to category of transmembrane adaptor proteins (TRAPs). They do not possess an enzymatic or kinase function, but they are involved in mediation of signal transmission from surface receptors to cell nucleus. We propose that some of them are engaged in development or maintenance of leukaemia.We have previously demonstrated that expression status of TRAPs at mRNA level is specific in some subgroups of childhood ALL, particularly in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) with the TEL/AML1 fusion gene. Furthermore, we have described that variable expression of NTAL mRNA is related to the response to initial glucocorticosteroid pre-phase in the treatment of childhood T-ALL; patients with high NTAL levels show better treatment response compared to the low-NTAL cases.In the current study, we aimed to prove experimentally that different levels of NTAL protein influence response of leukaemic T cells to glucocorticosteroids.In the wild type Jurkat cells (human T-cell leukaemia cell line) the NTAL protein is undetectable. For the in-vitro experiments we created a derivative Jurkat cell line transfected with the NTAL construct. In the derivative cell line, the NTAL positivity at both mRNA and protein level was verified using RT-PCR and Western blotting. The derivative cell line in a cell culture behaves similar as wild type Jurkat cell line. Transfectants and wild type Jurkat cells were incubated with methylprednisolone, dexamethasone or with solvent alone (H2O) as a negative control. Using flow cytometry we determined a percentage of surviving cells after 24, 48 and 72 hours of treatment. Cells were stained with Annexin V and DAPI and living cells were defined as Annexin V, DAPI negative. At each time point, the number of living cells in the negative control was set to 100%. After 48 hours of dexamethasone treatment the number of surviving cells in the Jurkat wild-type was higher by 12% compared to the Jurkat cells expressing NTAL. After 72 hours this difference was even more prominent reaching 46%. The same effect of methylprednisolone treatment was less pronounced (non-significant difference at 48 hours and 5% difference at 72 hours). Based on our experimental data we propose that NTAL acts in T-cells as a putative tumour suppressor. A plausible mechanism of its function is that NTAL competitively inhibits another adaptor LAT, which is required for signal propagation to the nucleus in T-cells. NTAL and LAT can compete for its localisation in proper “lipid rafts” or for palmitoylation. Our data can be used for subsequent functional analysis of signaling pathways in leukaemic blasts as well as in physiological lymphoid cells.

Nelarabine in combination with intensive modified BFM AALL00P2: A pilot study for the treatment of high risk T-ALL a report from the Children’s Oncology Group

10002 Background: Nelarabine has substantial clinical activity in relapsed T-ALL, but has been associated with significant neurotoxicity. Historically, children with high risk T-ALL, based on prednisone response or high-end induction MRD, have fared poorly with less than 50% cured with chemotherapy leading some groups to recommend allogeneic stem cell transplant in first remission for these patients. AALL00P2 was a two-stage pilot study designed to assess the feasibility and safety of adding Nelarabine to an intensive modified BFM regimen in children with newly-diagnosed T-ALL with high risk features. Methods: Ninety-two patients (89 eligible) were accrued from April 2001 to October 2005. In stage 1, patients with a slow early response (SER), defined as > 1000 peripheral blood blasts on day 8 of a prednisone prephase or MRD >1% at day 36 of induction therapy, were assigned to receive chemotherapy that included 5-day courses of Nelarabine at 400mg/m2/day; other patients received chemotherapy without Nelarabine. In stage 2, all patients received therapy with six courses of Nelarabine, at either 650mg/m2/day (SER patients, n=10) or 400mg/m2/day (others, n=51). Results: The 3-year EFS for stage 1 SER patients assigned to Nelarabine 400mg/m2 is 75% + 13% (n= 12), versus 75% + 11% for 16 good responders receiving no Nelarabine. Three-year EFS for all NCI high risk patients receiving Nelarabine 400mg/m2 (n=50) is 85%; 650mg/m2 (n=10) is 70%; and for no Nelarabine (n=13) 69%. There were no differences in either targeted or non-targeted toxicities among patients treated ± Nelarabine (Dunsmore et. al, Blood 108: 528a, 2006). Conclusions: In this pilot study, T-ALL patients with high-risk features and previously very poor outcomes, attained a 3-year EFS of >70% with intensive chemotherapy plus Nelarabine, without excess toxicity, and SER patients appeared to fare as well as good responding patients. These preliminary results have led the COG to randomize patients with T-ALL to intensive chemotherapy ± Nelarabine. AALL0434 will define the role of Nelarabine in patients with newly diagnosed T-ALL. No significant financial relationships to disclose.

Ability of Forage Grasses Exposed to Atrazine and Isoxaflutole to Reduce Nutrient Levels in Soils and Shallow Groundwater

Successful implementation of vegetative buffers requires inclusion of plant species that facilitate rapid dissipation of deposited contaminants before they have a chance to be transported in surface runoff or to shallow groundwater. Thirty‐six field lysimeters with six different ground covers [bare ground, orchardgrass (Dactylis glomerata L.), tall fescue (Festuca arundinacea Schreb.), smooth bromegrass (Bromus inermis Leyss.), timothy (Phleum pratense L.), and switchgrass (Panicum virgatum L.)] were established to evaluate the ability of grasses to reduce nutrient levels in soils and shallow groundwater. Nitrate (NO3 −) and orthophosphate (PO4 3−) were uniformly applied to each lysimeter. In addition, half of the lysimeters received an application of atrazine, and the other half received isoxaflutole (Balance™) at levels indicative of surface runoff from cropland. The leachate from each lysimeter was collected after major rainfall events during a 25‐day period, and soil was collected from each lysimeter at the end of the 25‐day period. Water samples were analyzed for NO3‐N and PO4‐P, and soil samples were analyzed for NO3‐N. Grass treatments reduced NO3‐N levels in leachate by 74.5 to 99.7% compared to the bare ground control, but timothy was significantly less effective at reducing NO3‐N leaching than the other grasses. Grass treatments reduced residual soil NO3‐N levels by 40.9 to 91.2% compared to the control, with tall fescue, smooth bromegrass, and switchgrass having the lowest residual levels. Switchgrass decreased PO4‐P leaching to the greatest extent, reducing it by 60.0 to 74.2% compared to the control. The ability of the forage grasses to reduce nutrient levels in soil or shallow groundwater were not significant between herbicide treatments. Quantification of microbial NO3 − dissipation rates in soil suggested that denitrification was greatest in switchgrass, smooth bromegrass, and tall fescue treatments. The overall performance of these three grasses indicated that they are the most suitable for use in vegetative buffers because of their superior ability to dissipate soil NO3 − and reduce nutrient transport to shallow groundwater.

Effect of Root Death and Decay on Dissipation of Polycyclic Aromatic Hydrocarbons in the Rhizosphere of Yellow Sweet Clover and Tall Fescue

A 12-mo greenhouse study was conducted to evaluate the contribution of root death and decay on the dissipation of polycyclic aromatic hydrocarbons (PAHs) in rhizosphere soil. The contaminated soil was previously treated by land-farming, but residual PAHs remained after treatment. Tall fescue (Festuca arundinacea Schreb.) and yellow sweet clover (Melilotus officinalis Lam.) were the target plants. To specifically evaluate the effect of root decay on contaminant dissipation, plants were treated with glyphosate, a broad spectrum herbicide, to induce root decay. Although tall fescue treatments had the highest root and shoot biomass and root surface area, this plant did not result in the highest contaminant degradation rates. Significant differences were noted between treatments for seven PAHs, with the active yellow sweet clover resulting in 60 to 75% degradation of these compounds. Induced root death and decay did not produce a significant enhancement of PAH degradation. The PAH microbial degrader populations in the vegetated treatments were more than 100 times greater than those in the unvegetated control. The phospholipid fatty acid (PLFA) structural group profile shifted over the growing period, indicating a change in the community structure. In conclusion, phytoremediation was shown to be an effective polishing tool for PAH-affected soil previously subjected to biological treatment.

MTOR as Therapeutic Target for T-Cell Leukemia: Synergism with Conventional Cytotoxic Drugs and Signaling Inhibitors.

Targeted disruption of signaling pathways essential for tumor cell survival and proliferation offer great promise for the design of more efficient and specific therapies. mTOR plays a central role in sensing mitogenic, nutrient and energy signals, and in the integration of different signaling pathways. Moreover, increasing evidence implicates mTOR in tumorigenesis and in the maintenance of transformed phenotypes. Therefore, we hypothesized that the mTOR pathway may play a significant role in T-cell acute lymphoblastic leukemia (ALL). BM and peripheral blood specimens from 15 pediatric patients were analyzed for the phosphorylation status of two downstream substrates of mTOR, S6 Ribosomal Protein and eIF4G. In all cases, primary leukemia T cells showed marked phosphorylation of S6 Ribosomal Protein (Ser235/236) and phosphorylation of the eukaryotic initiation factor eIF4G (Ser1108), showing that the mTOR pathway is constitutively active in T-ALL cells. Since leukemia T cells did not show significant basal phosphorylation of Akt/PKB, this observation suggests that the mTOR activation observed in T-ALL is Akt-independent. Specific blockade of mTOR by Rapamycin (100nM) reduced or abrogated phosphorylation of S6 ribosomal protein. Moreover, the mTOR blockers Rapamycin (10–100nM) and CCI-779 (10–100nM) significantly inhibited cell survival, and IL-7-promoted proliferation (40% to 85% inhibition) and cell cycle progression of T-ALL cells. Since we have shown previously that PI3K/Akt is critically involved in T-ALL responses to exogenous stimuli, we next evaluated whether inhibition of PI3K/Akt signals would synergize or potentiate the effects of mTOR blockade. We observed that the PI3K-inhibitor LY294002 (10μM) synergized with Rapamycin to abrogate IL-7-promoted leukemia T-cell proliferation. Finally, we explored the possibility that mTOR blockade can enhance the cytotoxic effects on T-ALL of the conventional drugs Dexamethasone (100nM) and Doxorubicin (100nM). Rapamycin potentiated the cytotoxic effects of both Dexamethasone (1.3 to 10 fold) and Doxorubicin (1.7 to 7.3 fold), suggesting that the combination of mTOR blockade with chemotherapy can improve the anti-leukemia efficacy of conventional regimens. In conclusion, we observed that the mTOR pathway is constitutively activated in primary leukemia T cells, and that the mTOR blockers Rapamycin and CCI-779 significantly inhibit T-ALL survival and proliferation. In addition, Rapamycin potentiates the inhibitory activity of other signaling inhibitors and the cytotoxic effects of the conventional drugs Dexamethasone and Doxorubicin. This study suggests that the mTOR pathway is a valid target for the treatment of T-ALL and lay the groundwork for the inclusion of mTOR blockade in the current therapeutic regimens.

Orchard Floor Management Influence on Summer Annual Weeds and Young Peach Tree Performance

This study compared the effect of weed control and orchard floor management (OFMA) options including organic mulch on summer annual weed interference in a newly established peach orchard. Weed interference where no preemergence (PRE) herbicides were applied, including vole damage, caused 29% peach tree mortality, reduced tree trunk cross-sectional area (TCSA) 62% by the fourth year of orchard establishment, and reduced fruit yield and fruit number in 1999 by 73 and 75%, respectively, but had no effect on fruit size. Compared with a no-till or conventionally tilled orchard floor, the population of grassy weeds within the tree row was greater in killed perennial ryegrass sod (PRS) plus hard fescue residue mulch treatments but was less in killed PRS plus tall fescue residue mulch treatments. Among the no-PRE treatments, the tree row broadleaf weed populations were suppressed in killed PRS with or without the addition of fescue residue mulch to the tree row when compared with the no-till or conventionally tilled orchard floor treatments. PRE herbicide treatments strongly affected peach fruit yield and TCSA but not average fruit size. There was no effect among the killed PRS, with or without hard or tall fescue residue mulch treatments, on peach fruit yield, TCSA, or average fruit size when compared with the no-till or conventionally tilled orchard floor treatment options. All treatments with herbicide had higher yields in 1999 than those without herbicides.

Effectiveness of Phytoremediation as a Secondary Treatment for Polycyclic Aromatic Hydrocarbons (PAHs) in Composted Soil

ABSTRACT A greenhouse study was conducted over a 12-month period to investigate the fate of polycyclic aromatic hydrocarbons (PAHs) in soil using phytoremediation as a secondary treatment. The soil was pretreated by composting for 12 weeks, then planted with tall fescue (Festuca arundinacea), annual ryegrass (Lolium multiflorum), and yellow sweet clover (Melilotus officinalis). Two sets of unvegetated controls also were evaluated, one fertilized and one unfertilized. Total PAH concentrations decreased in the tall fescue, annual ryegrass, and yellow sweet clover treatments by 23.9%, 15.3%, and 9.1%, respectively, whereas the control was reduced by less than 5%. The smaller two- and most of the three-ringed compounds—naphthalene, acenaphthylene, acenaphthene, fluorene, and anthracene—were not found in detectable concentrations in any of the treatments. The most probable number analysis for microbial PAH degraders did not show any statistically significant differences among treatments. There were significant differences among treatments (p < 0.05) for the residual concentrations of five of the target PAHs. Root surface area measurements indicated that tall fescue and annual ryegrass both had significantly higher root surface area than yellow sweet clover, although the two species were not significantly different from each other. The tall fescue treatment resulted in the highest root and shoot biomass, followed by annual ryegrass and yellow sweet clover, and also had the highest percent of contaminant removal after 12 months. These results imply a positive relationship between plant biomass development and PAH biodegradation.

The effect of grazing by cattle on animal performance and floristic composition in Nardus‐dominated swards

Four groups of six spring‐calving beef cows and their calves were allocated to two replicated grazing treatments on a Nardus stricta‐dominated hill pasture in the UK during six consecutive summer grazing seasons. Treatments were applied by continuous variable stocking to maintain a sward height of either 4–5 cm (short) or 6–7 cm (tall) between tussocks of N. stricta.Cows on both treatments ingested a greater proportion of N. stricta than was present in the sward. The short treatment reduced the herbage intake of cows proportionally by 0·35 (P &lt; 0·001) and reduced herbage digestibility by 0·03 (P &lt; 0·01) compared with the tall treatment. Cows lost 0·02 kg d−1 live weight on the short treatment but gained 0·27 kg d−1 live weight on the tall treatment (s.e.d. 0·077; P &lt; 0·001). Calf liveweight gains were 0·60 and 0·86 (s.e.d. 0·034; P &lt; 0·001) kg d−1 for the short and tall treatments respectively. The greatest liveweight gains for both cows and calves occurred during the first 5–6 weeks of the grazing season and differences between treatments increased over the season. Cattle on the short treatment consistently grazed a higher proportion of N. stricta tillers more closely than those on the tall treatment, and the percentage cover of N. stricta declined more rapidly on the short treatment. After 6 years of grazing there was a considerably greater (P &lt; 0·001) proportion of very small tussocks, i.e. less than 140 cm2 basal area, on the short treatment than on the tall treatment. The percentage cover of broad‐ and fine‐leaved grasses also declined, in contrast to previous studies. This may have been due to lower soil fertility on the site of the current experiment.It is concluded that high levels of utilization of N. stricta are incompatible with high levels of individual animal performance in lactating cows, although short periods in early summer may give acceptable performance. Lower levels of utilization such as that achieved on the 6–7 cm treatment resulted in moderate levels of animal performance and also gradually reduced the cover of N. stricta, although it took longer for the effect to be established.

Multidrug Resistance in Aggressive Lymphoproliferative Disorders of T and Natural-Killer Origin

The measurement of rhodamine 123 (Rho123) efflux in hematological malignancies, using flow-cytometry, provides an accurate assessment of multidrug resistance (MDR) of both P-glycoprotein and MRP. While their normal counterparts display high levels of PgP and Rho123 efflux, we investigated the MDR status of marked T/NK proliferations. When diagnosed according to natural killer (NK) markers (CD16, CD56, CD57) 8 of nine NK lymphoproliferative disorders (LPD) were markely positive (3 NK non Hodgkin's lymphomas (NHL), 1 NK lymphoproliferative disease of large granular lymphocytes (LGL), and 5 T/NK LGL). These results are in accordance with the observed response to chemotherapy in the treated cases. Mature T LPD (prolymphocytic leukemia (PLL), and NHL) cells gave varying results, as did cells from Sezary syndromes. Marked Rho123 efflux was detected in the two cases of T-PLL suggesting the expression of MRP as previously described. Immature T-lymphomas or leukemias (6 cases) were all negative. These data should be considered in relation to NK proliferations which clearly display an MDR phenotype and therefore raise the question, of the relevance of this phenotype in normal cells, and secondly of the negativity of immature T-LPD. The latter could indicate that MDR inhibitors may be superfluous in the initial treatment of acute lymphoblastic leukemia (ALL). Finally the resistance to treatment of T-ALL or mature T cells LPD invokes the importance of exploring other mechanisms of drug resistance such as the lung resistance related protein (LRP).