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Abstract 11 Glucocorticoids (GC) play a fundamental role in the treatment of all lymphoid tumors because of their capacity to induce apoptosis in lymphoid progenitor cells. The importance of GC therapy in lymphoid malignancies is underscored by the strong association of GC response with prognosis in childhood acute lymphoblastic leukemia (ALL). Thus, resistance to glucocorticoids is a well recognized feature of poor prognosis in the treatment of childhood ALL. A number of different mechanisms contributing to GC resistance in T-ALL have been proposed including increased expression of antiapoptotic factors such as MCL1, insufficient expression of the glucocorticoid receptor gene (NR3C1), expression of NR3C1 splice variants and loss of NR3C1 auto up-regulation. The development of strategies to reverse glucocorticoid resistance could have a profound impact on the treatment of ALL. Recently, we have showed that inhibition of NOTCH1 with gamma secretase inhibitors (GSIs) sensitized GC-resistant T-ALL cell lines and primary samples to GC-induced apoptosis, supporting a role for GC plus GSIs in the treatment of GC-resistant T-ALLs. Here, we have used a Systems Biology approach to uncover additional pathways involved in the regulation of glucocorticoid receptor activity and glucocorticoid resistance in ALL. In this approach we used Master Regulator Inference analysis (MaRInA), a novel algorithm designed to identify critical regulators of complex biological traits, to analyze the gene expression profiles of pre-treatment T-ALL samples determined to be either sensitive (IC50150μ g/ml) to GC-induced apoptosis in vitro. This analysis identified a transcriptional module controlled by the AKT pathway as the most highly enriched gene set in the resistant samples, strongly suggesting a critical role for PI3K-AKT signaling in the regulation of glucocorticoid resistance in T-ALL. Notably, loss PTEN and constitutive activation of AKT are highly prevalent in T-ALL. Analysis of the effects of AKT in the transcriptional activity of the glucocorticoid receptor and in glucocorticoid receptor induced apoptosis revealed that constitutive activation of AKT results in impaired glucocorticoid receptor activity. Based on these results we hypothesize that AKT could have a direct inhibitory effect on the glucocorticoid receptor. Consistent with this hypothesis, mass spectrometry analysis demonstrated high levels of phosphorylation of the NR3C1 Ser134 in cells harbouring constitutively active AKT. Notably, this residue is within an AKT consensus sequence, XRXXS, and is highly conserved amongst species. Moreover, protein pull down and in vitro kinase assays demonstrated that AKT can directly interact with NR3C1 and mediates its phosphorylation on Ser134. Analysis of the biological relevance of this posttranslational modification showed that constitutively active AKT induces: (i) marked decrease in NR3C1 stability; (ii) retention of NR3C1 in the cytosol in the presence of dexamethasone and (iii) impaired glucocorticoid receptor autoupregulation; all of which can be blocked by the expression of a phosphorylation-deficient serine to alanine (S134A) NR3C1 mutant. Analysis of the effects of AKT activation on the interaction between NOTCH1 signaling and glucocorticoid induced apoptosis showed that AKT can effectively block the reversal of glucocorticoid resistance induced by inhibition of NOTCH1 signaling with GSIs in a Ser134 phosphorylation dependent manner. Finally, we demonstrate that pharmacologic inhibition of AKT can effectively reverse glucocorticoid resistance in T-ALL primary human samples and cell lines. Overall these results identify a direct interaction between the PI3K/AKT pathway and NR3C1 signaling and provide a strong rationale for the clinical testing the combination of AKT inhibitors and glucocorticoids in T-ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 141 Introduction: T-cell lymphoblastic leukemia (T-ALL) arises from clonal expansion of a lymphoid progenitor that has undergone stepwise alteration at distinct stages of differentiation. It is suggested that a set of cooperative mutations that affect different pathways are required before thymocytes become fully malignant. Despite major improvements in our understanding of the molecular genetics of T-ALL, the underlying mechanisms that lead to the abnormal proliferation and enhanced survival of the leukemic cells remain largely unknown. Results: Array CGH analysis revealed an acquired homozygous microdeletion at chromosome 18p11 in 6 % of T-ALL cases. The deleted region was only 125 kb in size and restricted to the PTPN2 (protein tyrosine phosphatase, non-receptor type 2) locus. PTPN2 encodes an intracellular non-transmembrane tyrosine-specific phosphatase that functions as a negative regulator of a variety of signaling proteins including several members of the janus kinase (JAK) and of signal transducer and activator of transcription (STAT) families, growth factor receptors and SRC family kinases. Homozygous deletion of PTPN2 was specifically found in cases with aberrant expression of the TLX1 transcription factor, with two cases also harboring the NUP214-ABL1 fusion. Analysis of additional TLX1 positive cases by quantitative PCR identified loss of one copy of PTPN2 in 5 out of 20 cases. No mutations were detected in the coding region of PTPN2. To determine the effect of loss of PTPN2 in T-cells, we downregulated the expression of PTPN2 using RNAi technology. siRNA mediated knock-down of PTPN2 affected activation of JAK1 associated cytokine receptors implicated in T-cell development. Ligand stimulation of IL7 and interferon gamma receptor resulted in an augmented and prolonged phosphorylation of JAK1 as well as downstream targets STAT1 and STAT5 in T-ALL cell lines with knock-down of PTPN2. In addition, knock- down of Ptpn2 sensitized the pro B-cell line Ba/F3 to transformation by wild type JAK1 confirming a clear relationship between loss of PTPN2 and JAK1 activation. Knock-down of PTPN2 expression also provided a proliferative advantage and reduced sensitivity to kinase inhibitors in lymphoblastic leukemia cell lines HSB-2 and ALL-SIL. Conclusion: In conclusion, our data provide genetic and functional evidence for a tumor suppressor role of PTPN2 in T-ALL and warrant testing of JAK inhibitors for the treatment of this specific subset of T-ALLs as well as further analysis of a potential negative impact of loss of PTPN2 on responsiveness to anti-cancer treatments. Disclosures: Ferrando: Merck, Pfizer: Research Funding.

Abstract 3588 Poster Board III-525 The molecular pathways regulating mast cell (MC) development in vertebrates remain to be elucidated. The Notch signaling pathway is highly conserved in all metazoans and has been implicated in regulating hematopoietic stem cell induction and lineage cell fate decisions. Notch receptors and their ligands are expressed in a number of hematopoietic cells, including MCs. We were the first to identify zebrafish MC equivalents (Dobson et al., Blood 2008) and examine vertebrate MC transcriptional regulation in vivo. These studies demonstrated the significance of carboxypeptidase A 5 (cpa5) as a zebrafish MC-specific marker. Co-localization studies reveal zebrafish notch3 (a homologue of human NOTCH3) is expressed in a proportion of cpa5 positive cells in 7 day old embryos. Moreover, the zebrafish Notch signaling mutant, mind bomb, displays profound loss of cpa-5 expression, as do wild type zebrafish embryos treated with Compound E (Cpd E), a gamma-secretase inhibitor that blocks Notch signaling. We previously identified pu.1 and gata2 as essential transcription factors for early MC development. Interestingly, we observed a dose-dependent response, with reduced cpa5 and gata2 but preserved pu.1 expression at 50 μM Cpd E, compared with profound decreased expression of all these factors, as well as gata1 and mpo at 75 μM Cpd E. These data suggest a particular role for Notch signaling in regulating MC development, as well as a potentially broader role in regulating the myeloid and erythroid lineages. These studies are currently being validated through reciprocal experiments overexpressing notch mRNA in wild type embryos and rescue experiments overexpressing the notch intracellular domain and the above-mentioned transcription factors in Notch deficient embryos (mind bomb and Cpd E treated). We have also developed a transgenic zebrafish line expressing the human c-KIT D816V mutation found in systemic mastocytosis, which exhibits increased mast cells at the expense of erythroid cells, features in keeping with the human condition. These transgenic fish provide an opportunity to examine if Notch pathway inhibition alone, or in combination with other therapies, such as those targeting the c-KIT kinase, have a therapeutic impact in this condition. Parallel approaches in a human mastocytosis cell line are also being undertaken. These studies promise key insight into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model for identifying novel therapeutic strategies in MC diseases. Disclosures: Ferrando: Merck, Pfizer: Research Funding.

Abstract 676 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of transcription factor oncogenes. TLX1/HOX11 was originally isolated from the recurrent t(10;14)(q24;q11) translocation and is aberrantly expressed in 5% to 10% of pediatric and up to 30% of adult T-ALL. The TLX3/HOX11L2 oncogene is closely related to TLX1 and is activated by the cryptic t(5;14)(q35;q32) chromosomal translocation in 25% of pediatric T-ALLs. TLX1 and TLX3 positive T-ALLs have a distinct gene expression profile resembling that of thymocytes blocked at the early double-positive stage of development, suggesting that aberrant expression of TLX1 and TLX3 interferes with critical transcriptional regulatory networks that control cell proliferation, differentiation and survival during T-cell development. However, the identity of such oncogenic pathways and the mechanisms through which they operate are still largely unknown. Here we used an integrated systems biology approach to elucidate the transcriptional regulatory networks controlled by aberrant expression of TLX1 and TLX3 in T-ALL. Towards this goal, we performed gene expression profiling studies and ChIP-on-chip analysis of TLX1 and TLX3 direct target genes in leukemic lymphoblasts. ChIP-on-chip analysis of TLX1 direct target genes identified 5,550 promoters bound by TLX1 in ALL-SIL T-ALL cells with a significance cutoff of P 75% overlap with the TLX3 ChIP-on-chip target genes (P Disclosures: No relevant conflicts of interest to declare.

Abstract 366 Gain of function mutations in Notch1, which encodes a signaling protein that is converted into a transcription factor upon activation, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Although inhibiting Notch1 activity represents a potential therapeutic opportunity, the discovery of new Notch1 pathway antagonists poses a difficult challenge. Traditional small molecule library screening approaches have not been amenable to modulating transcription factor abnormalities. In order to overcome this challenge, we applied Gene Expression-based High-throughput Screening (GE-HTS) to discover new Notch1 modulators. GE-HTS uses gene expression signatures as surrogates for different biological states. We derived a 32-gene Notch1 expression signature from genome-wide microarray expression profiling of 7 different Notch1 mutant T-ALL cell lines treated with vehicle (Notch1 on) versus a Notch1 inactivating γ-secretase inhibitor (GSI; Notch off) and screened a small molecule library for compounds inducing the Notch1 off state in DND41 mutant Notch1 T-ALL cells. Among numerous ion flux modulators validated to induce the Notch1 off signature, one of the top hits was the FDA-approved calcium channel blocker, bepridil, used to treat patients with cardiac disease. In multiple mutant Notch1 T-ALL cell lines, bepridil induced the Notch1 off signature. We next confirmed that bepridil indeed targets Notch signaling by demonstrating its inhibitory effects on a Notch-sensitive luciferase reporter gene in heterologous U2OS cells expressing a mutated form of Notch1. Similar to the phenotypic effects of GSI, bepridil induced a G0/G1 cell cycle arrest, inhibited cellular viability, and decreased cell size in multiple T-ALL cell lines, including the GSI-resistant cell line PF382. Next, in order to confirm dependency of the induced phenotype on inhibition of Notch, we utilized the 8946 T-ALL cell line. This murine line depends on a doxycycline-repressible human c-myc transgene for growth and can be rescued from transgene withdrawal with activated Notch1, which upregulates the endogenous c-myc gene. In these cells, the phenotypic effect of bepridil on viability is also dependent on Notch1 inhibition because cells rescued from transgene withdrawal with activated Notch1 were more sensitive to the effects of the drug than were those cells still dependent on the c-myc transgene. Finally, we asked whether bepridil altered the level of active Notch1 protein in T-ALL cell lines. As with GSI, bepridil treatment results in decreased levels of intracellular Notch (ICN1). In contrast to GSI, however, bepridil treatment decreased levels of the furin-processed extracellular and transmembrane forms of Notch1 while the full length Notch1 precursor form accumulated upon bepridil treatment. One hypothesis is that by altering ER/Golgi compartment calcium, bepridil prevents the folding of newly synthesized Notch1 polypeptides, leading to its retention in the ER/Golgi and a failure to traffic to cellular compartments where receptor activation occurs. Consistent with this hypothesis co-localization studies in U2OS cell lines expressing the L1601P mutant Notch1 suggest retention of Notch1 in the ER/Golgi. An alternative hypothesis under investigation is that bepridil affects the activity of furin, a calcium-dependent protease that is required for processing of Notch receptors. In summary, we have identified an FDA-approved drug with Notch1 modulating activity in T-ALL by a mechanism unique from GSI. These studies have potential for rapid translation to clinical testing. Disclosures: Ferrando: Merck, Pfizer: Research Funding.

Abstract 142 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of transcription factor oncogenes. TLX1/HOX11 was originally isolated from the recurrent t(10;14)(q24;q11) in T-ALL and is aberrantly expressed in 5% to 10% of pediatric and up to 30% of adult T-ALL cases. Tlx1 plays an important role during embryonic development and acts as a master transcriptional regulator necessary for the genesis of the spleen. TLX1 positive T-ALLs have a distinct gene expression profile resembling that of thymocytes blocked at the early double positive stage of development. This observation supports the hypothesis that aberrant expression of TLX1 contributes to the pathogenesis of T-ALL by interfering with critical transcriptional regulatory networks involved in cell proliferation, differentiation and survival during T-cell development. However, the identity of such oncogenic pathways and the mechanisms though which they operate are still largely unknown. In order to gain further insight into the mechanisms of transformation induced by TLX1, we generated a transgenic model of TLX1 induced T-ALL. In this model, a human TLX1 cDNA was expressed in developing thymocytes under the control of the proximal LCK promoter. TLX1 transgenic mice displayed a specific defect in T-cell development characterized by reduced thymic size and cellularity with increased apoptosis and a differentiation arrest at the CD4/CD8 double negative, CD25/CD44 double positive stage of differentiation (DN2 thymocytes). Long term follow up revealed that TLX1 transgenic mice develop tumors with a median latency of 29 weeks. TLX1 induced leukemias are characterized by increased thymic size and diffuse infiltration of bone marrow, spleen and peripheral organs by lymphoblasts expressing T-cell markers. Analysis of TCRβ expression and transplantation into isogenic recipients demonstrated that TLX1 tumors are clonal and transplantable. Microarray gene expression profiling of mouse and human T-ALLs showed that tumors from TLX1 transgenic mice have a gene expression signature that is highly related to that of human T-ALLs with aberrant expression of TLX1. Moreover, ChIP-on-chip analysis of promoters bound by TLX1 showed this genetic program is dominated by the downregulation of TLX1 direct target genes. Mutation profiling of T-cell oncogenes and array CGH analysis in mouse TLX1 tumors demonstrated the presence of cooperative mutations including Pten deletions, activating mutations in Notch1 and loss of Bcl11b. Strikingly, SKY analysis and array CGH revealed that 80% of TLX1 induced tumors had numerical chromosomal abnormalities including a high frequency of trisomy 15. Analysis of gene expression of ChIP-on-chip TLX1 direct target genes in double negative thymocytes from preleukemic mice showed downregulation of genes primarily involved in the control of chromosomal segregation during mitosis such as Bub1, Brca2, Chek1, Kntc1, Kif23, CenpE and Plk1. These data suggest that aberrant TLX1 expression directly promotes aneuploidy and contributes to T-cell transformation by interfering with the expression of genes responsible for chromosomal segregation during mitosis. Importantly, T-cell lymphoblasts from TLX1 transgenic mice failed to undergo a mitotic cell cycle arrest after treatment with taxol, a cell cycle inhibitor that interferes with microtubule remodeling during mitosis. Strikingly, karyotype analysis of a series of 59 pediatric T-ALLs demonstrated a high frequency of chromosomal gains and losses in TLX1 and TLX3 positive human T-cell tumors compared with other genetic subgroups of T-ALL (P Disclosures: Ferrando: Merck, Pfizer: Research Funding.

Notch1 activation is essential for T-lineage specification of lymphomyeloid progenitors seeding the thymus. Progression along the T cell lineage further requires cooperative signaling provided by the interleukin 7 receptor (IL-7R), but the molecular mechanisms responsible for the dynamic and lineage-specific regulation of IL-7R during thymopoiesis are unknown. We show that active Notch1 binds to a conserved CSL-binding site in the human IL7R gene promoter and critically regulates IL7R transcription and IL-7R  chain (IL-7R) expression via the CSL–MAML complex. Defective Notch1 signaling selectively impaired IL-7R expression in T-lineage cells, but not B-lineage cells, and resulted in a compromised expansion of early human developing thymocytes, which was rescued upon ectopic IL-7R expression. The pathological implications of these findings are demonstrated by the regulation of IL-7R expression downstream of Notch1 in T cell leukemias. Thus, Notch1 controls early T cell development, in part by regulating the stage- and lineage-specific expression of IL-7R., This work was supported by grants from Plan Nacional (SAF2004-01122 and BFU 2007-60990), Comunidad de Madrid (S-SAL0304-2006), Fundación la Caixa (ON03/ 109-00), Fundación MM, and Instituto de Salud Carlos III (RECAVA RD06/0014/1012 and RD06/0014/0038 to M.L. Toribio and J.L. de la Pompa, respectively), and by an Institutional Grant from the Fundación Ramón Areces. S. González-García was supported by Ministerio de Ciencia e Innovación (MICINN; FPI program), M. García-Peydró by CSIC (I3P program),and E. Martín-Gayo by MICINN (FPU program) and by CAM.

A risk-adapted approach to the treatment of patients with acute lymphoblastic leukemia (ALL) has the potential of improving survival in high-risk patients and reducing therapy-related long-term sequelae in those at low-risk. The use of molecular markers that can further define risk are needed in this disease. Activating mutations of Notch-1 have been reported in over 50% of patients with T-ALL. Mutations in the heterodimerization domain (HD) lead to ligand-independent cleavage, whilst truncating mutations of the C-terminal PEST domain disrupt binding to the negative regulator FBXW7, leading to prolonged half-life of intracellular Notch (ICN). More recently, Notch-activating mutations in FBXW7 that prevent ICN degradation have been reported in 20% of patients. In pediatric patients treated on the ALL-BFM 2000 protocol, patients with Notch-1 mutations (MUT) had an excellent outcome with an event free survival (EFS) of 90% compared to 71% in wild type (WT) patients. The impact of FBXW7 mutations was not evaluated. We sought to address whether adults with Notch-1 and/or FBXW7 mutations also faired sufficiently well that they might avoid treatment intensification/transplantation. Notch-1 HD-N, HD-C, TAD and PEST domains, and FBXW7 WD40 domain were screened by denaturing high performance liquid chromatography and/or bidirectional sequencing in a cohort of 88 adult T-ALL patients treated on the MRC UKALLXII/ECOG2993 trial (54 UKALLXII, 34 ECOG2993). Overall, 51 patients (58%) had at least one Notch-1 mutation; 37 in the HD only, 8 in the PEST domain only and 6 in both HD and PEST domains. Sixteen patients (18%) had an FBXW7 mutation (7 R465C, 3 R505C, 2 R479Q, 2 R479L, 1 R465H, and 1 G423V) and, of note, 15 of these mutations altered conserved arginine residues in the WD40 domain thought to be responsible for binding to the Notch-1 PEST domain. Five FBXW7 MUT patients were Notch-1 WT. There was a positive association between having an FBXW7 mutation and a Notch-1 mutation in the HD only (Fishers exact test 2P.02). Furthermore, no patient had both an FBXW7 and a PEST mutation. This is consistent with the hypothesis that Notch-1 HD and FBXW7 mutations act in concert similar to dual HD and PEST mutations. Complete remission was achieved in 96% of patients. Median follow-up was 3.5 years. The Notch-1 WT and MUT groups received similar treatment (8 Vs 15 sibling allograft; 3 Vs 7 autograft; 3 Vs 3 matched unrelated donor allograft; 19 Vs 24 chemotherapy with maintenance alone; P.84). There was no significant association with white cell count (WCC) or age according to either Notch-1 or FBXW7 mutational status (Mantel Haenszel test for trend P>0.1 for each case). Outcome is shown in table 1. In contrast to the data in pediatric T-ALL, in this adult cohort Notch-1 mutational status was not associated with improved EFS or overall survival (OS); nor was there an association according to the type of Notch-1 mutation. Similarly, FBXW7 mutational status alone was not predictive of outcome. There was a suggestion that patients with either a Notch and/or an FBXW7 mutation had better EFS, although this did not reach statistical significance. Adjusting for treatment received in first remission did not affect the results. In conclusion, the outcome of adult patients remains poor irrespective of their Notch-1 and/or FBXW7 mutation status, and there is no evidence that these markers should influence therapy in this age group. 5 yr EFS (WT v MUT) OR (CI) log rank 2p Notch 37% v 47% 0.79 (0.44–1.42) 0.4 FBXW7 41% v 53% 0.85 (0.40–1.82) 0.7 NOTCH +/− FBXW7 31% v 49% 0.70 (0.38–1.29) 0.3 5 yr OS WT v MUT) OR (CI) log rank 2p Notch 47% v 51% 0.87 (0.47–1.62) 0.7 FBXW7 46% v 62% 0.76 (0.35–1.66) 0.5 NOTCH +/− FBXW7 44% v 52% 0.82 (0.43–1.56) 0.5

Activating mutations in NOTCH1 are present in over 50% of T-cell acute lymphoblastic leukemias (T-ALL). Consequently, inhibition of NOTCH1 signaling with small molecule γ-secretase inhibitors (GSIs) has been proposed as a targeted therapy for this disease. However, GSIs fail to induce robust apoptosis in T-ALL cells and their clinical application has been limited by the development of severe gastrointestinal toxicity (mucous diarrhea and goblet cell metaplasia) resulting from systemic inhibition of NOTCH signaling. Here we show that combination therapy with GSIs plus glucocorticoids can reverse both glucocorticoid resistance in T-ALL and the occurrence of GSI-induced gut toxicity in vivo. The development of glucocorticoid resistance in T-ALL has been associated with the loss of a positive auto-regulatory transcriptional feedback loop required for glucocorticoid induced apoptosis. Using gene expression profiling and ChIP-on-chip analysis we demonstrated that HES1, a transcriptional repressor controlled by NOTCH1, inhibits specific regulatory elements in the glucocorticoid receptor promoter required for effective glucocorticoid receptor autoupregulation and glucocorticoid induced cell death. Consequently, downregulation of HES1 via inhibition of NOTCH1 signaling with GSIs or HES1 inactivation via shRNA knockdown restores glucocorticoid receptor autoupregulation and reverses glucocorticoid resistance in T-ALL cell lines and primary patient samples. Consistent with these results, combination therapy with dexamethasone and GSIs was highly effective in a mouse xenograft model of glucocorticoid resistant T-ALL. Surprisingly, these studies demonstrated not only improved antileukemic responses in mice treated with dexamethasone plus a GSI compared with dexamethasone or placebo treated controls (P

Although in recent years intensive chemotherapy regimens have improved outcome for patients with T-cell acute lymphoblastic leukemia (T-ALL), side effects and relapse-rates remain high. An alternative therapeutic approach would be to target constitutively active mutated Notch1. Notch1, a trans-membrane receptor that acts as a transcription factor following proteolytic activation by the γ-secretase complex, harbors gain of function mutations in over 50% of T-ALL. Early clinical trials attempting to modulate Notch1 with γ-secretase inhibitors (GSI) were closed secondary to gastrointestinal toxicity, suggesting that alternative approaches to inhibiting Notch1 signaling are needed. Transcription factor complexes are difficult to target pharmacologically with conventional high-throughput screening approaches. We therefore sought to target Notch1 with a new gene expressionbased approach to small molecule library screening. Signatures for the Notch1 on versus off states were defined using microarray expression profiling of 7 different Notch1 mutant T-ALL cell lines treated with vehicle (Notch1 on) versus a GSI (Notch1 off). We adapted this 32-gene signature to our gene expression-based high-throughput screening (GE-HTS) assay, which uses ligation-mediated amplification (LMA) and a Luminex bead-based detection system. As proof of principle, we confirmed that an shRNA against Notch1 induced the Notch1 off signature in DND41 cells (a T-ALL cell line bearing a mutated Notch1 allele). In order to facilitate future clinical translation, we screened a collection of 4,500 bioactive compounds highly enriched for FDA-approved drugs in DND41 cells. Twenty-eight compounds that induced the Notch1 off signature were identified as top hits. These compounds were tested in a secondary screen in a 2-fold dilution series in two GSI sensitive cell lines, DND41 and KOPTK1, and one GSI resistant cell line, PF382. Ion channel modulators emerged as the top class of compounds confirmed to induce the Notch1 off signature across all cell lines. We next focused on two representative compounds, the ionophores salinomycin and ionomycin. Similar to GSI, both of these molecules induced cell cycle arrest and markedly decreased levels of the activated intracellular portion of Notch (ICN). Both salinomycin and ionomycin, as well as numerous other ionophores that scored as inhibitors in the screen, are known to increase cytosolic calcium. These studies are consistent with a role for calcium in the activation of oncogenic forms of Notch1 and suggest a potential role for ion channel modulators in the treatment of T-ALL.

Recent studies have shown that activating mutations of NOTCH1 are responsible for the majority of T cell acute lymphoblastic leukemia (T-ALL) cases. Most of these mutations truncate its C-terminal domain, a region that is important for the NOTCH1 proteasome-mediated degradation. We report that the E3 ligase FBW7 targets NOTCH1 for ubiquitination and degradation. Our studies map in detail the amino acid degron sequence required for NOTCH1-FBW7 interaction. Furthermore, we identify inactivating FBW7 mutations in a large fraction of human T-ALL lines and primary leukemias. These mutations abrogate the binding of FBW7 not only to NOTCH1 but also to the two other characterized targets, c-Myc and cyclin E. The majority of the FBW7 mutations were present during relapse, and they were associated with NOTCH1 HD mutations. Interestingly, most of the T-ALL lines harboring FBW7 mutations were resistant to γ-secretase inhibitor treatment and this resistance appeared to be related to the stabilization of the c-Myc protein. Our data suggest that FBW7 is a novel tumor suppressor in T cell leukemia, and implicate the loss of FBW7 function as a potential mechanism of drug resistance in T-ALL.

Gamma-secretase inhibitors (GSIs), which block the activation of the NOTCH1 receptor, are currently being tested in the treatment of T-cell lymphoblastic leukemias (T-ALL) with activating mutations in the NOTCH1 gene. However, inhibition of NOTCH1 signaling induces only a delayed cytostatic antileukemic effect with little or no apoptosis and GSI treatment is associated with severe gastrointestinal toxicity, which limits the clinical application of these molecularly targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids reverses glucocorticoid resistance and is highly effective against T-ALL. Inhibition of NOTCH1 signaling with CompE, a highly active GSI, in combination with dexamethasone, induced apoptotic cell death in glucocorticoid-resistant T-ALL cell lines. The synergistic antileukemic effect of GSIs and glucocorticoids was effectively rescued by forced expression of ICN1, an intracellular form of NOTCH1 that does not require gamma-secretase processing; or by treatment with the glucocorticoid receptor antagonist RU486. Reversal of glucocorticoid resistance by CompE treatment was mediated by transcriptional upregulation of BIM, a BH3-only proapototic factor previously involved in glucocorticoid induced cell death. Thus, shRNA knock down of BIM effectively impaired the ability of CompE plus dexamethasone to induce programmed cell death in leukemic lymphoblasts. Using a mouse xenograft model of glucocorticoid-resistant T-ALL we demonstrated that the combination of DBZ, a GSI with well established activity in vivo, and dexamethasone is highly effective against glucocorticoid resistant T-ALL in vivo. All animals treated with DBZ plus dexamethasone showed complete clearance of leukemic lymphoblasts and improved survival, while mice treated with vehicle or dexamethasone alone died because of disease progression (P

Aberrant activation of NOTCH1 signaling induces transformation of T-cell progenitors and plays a prominent role in the pathogenesis of over 50% of human T-cell acute lymphoblastic leukemias (T-ALL), which harbor activating mutations in the heterodimerization (HD) and PEST domains of NOTCH1. Here we report a new class of activating mutations in NOTCH1 in human T-ALL. These so called JuxtaMembrane Expansion (JME) mutants consist of internal tandem duplications of exon 28 and adjacent intronic sequences in the NOTCH1 gene, which result in expansions of the extracellular juxtamembrane region of the NOTCH1 receptor. Western blot analysis of T-ALL cell lines lacking known NOTCH1 mutations demonstrated high levels of activated NOTCH1 protein in Jurkat T-ALL cells, suggesting the presence of an as yet unidentified activating NOTCH1 mutation in this cell line. Sequence analysis of Jurkat NOTCH1 transcripts revealed an internal tandem duplication in exon 28 of NOTCH1, resulting in the insertion of 17 amino acids at position 1740 in the extracelullar juxtamembrane region of the receptor. Subsequent PCR amplification of NOTCH1 exon 28 sequences from 194 primary T-ALL lymphoblast samples identified seven additional in frame insertion mutations ranging from 11 to 36 amino acids in length, all of which were located in the vicinity of codon 1740 in the extracelullar juxtamembrane region of the NOTCH1 receptor. Luciferase assays showed that expression of the NOTCH1 Jurkat JME17 mutant allele induced over 200 fold activation of a NOTCH1 reporter construct compared to controls. Activation of NOTCH1 signaling requires proteolytic cleavage of the receptor, first by an ADAM metalloprotease (S2 clevage) and subsequently by the gamma-secretase complex. NOTCH1 signaling induced by the Jurkat JME17 mutant was completely abrogated by incubation with CompE, a highly active gamma-secretase inhibitor. Consistently, treatment of Jurkat T-ALL cells with CompE resulted in rapid clearance of activated NOTCH1 protein and in marked downregulation of NOTCH1 target genes such as HES1 and DELTEX1. Interestingly, and in contrast with previously described HD mutations, JME NOTCH1 alleles retain an intact HD domain and a protected canonical S2 metalloprotease cleavage site. Thus, we hypothesized that activation of NOTCH1 by JME mutations could be mediated by aberrant metalloprotease cleavage at ectopic S2 sites within the JME insertion sequence. However, mutation of the canonical S2 cleavage abrogated the function of the NOTCH1 Jurkat JME17 mutant allele. Furthermore, analysis of artificially generated JME insertions containing sequences unrelated to the leukemia-derived JME alleles, showed that activation of NOTCH1 by JME mutations depends primarily on the length of the inserted peptides and not on their specific amino acid sequences. Thus, shorter insertions of 5 to 13 amino acids in length induced moderate (5–10 fold) activation of the NOTCH1 receptor, while insertions of 14 amino acids or longer induced marked (>70 fold) increases in NOTCH1 signaling. Overall, these results provide further insight in the mechanisms that control the activation of the NOTCH1 receptor in T-ALL.

Transgenic mouse models of T-cell oncogenes such as TAL1 and LMO1, have confirmed their critical role in the development of T-lymphoblastic lymphoma (T-LL). In addition, retroviral insertional mutagenesis (RIM) in mice is a powerful system for the identification of genes involved in T-cells leukemogenesis. However, incomplete knowledge of the pathogenesis of T-ALL limits the ability to stratify patients and to deliver tailored therapy accordingly. Thus, a major goal is to identify common oncogenic pathways downstream of T-cell oncogenes that can serve as therapeutic targets for the treatment of T-ALL. We hypothesize that T-cell oncogenes operate through a limited number of oncogenic pathways with distinct gene expression profiles. Furthermore, we propose that gene expression profiling of murine models of T-cell lymphomas, which harbor specific genetic lesions, will serve to identify such oncogenic pathways and to establish a molecular classification of T-ALL. To test this hypothesis we have analyzed normal thymus and mouse T-cell lymphomas originated from retroviral insertional mutagenesis (n=11) and transgenic and knock-outs models (n=30) using Affymetrix 430A2.0 microarrays. Unsupervised analysis clearly distinguishes tumor samples from normal thymus and clusters the tumors into three major groups. TAL1, LMO2 and E2 proteins are known to be part of a transcriptional complex and to cooperate in T-cell leukemogenesis by suppressing E2A function. Accordingly, mouse tumors originating in TAL1, TAL1/LMO2 transgenic and E2A knock-out mice share a common gene expression signature and cluster together. Supervised analysis of tumors generated by retroviral mutagenesis showed increased expression of the proviral tagged genes and identified corresponding known downstream targets. Nearest neighbor analysis identified high levels of Notch1 expression in tumors with proviral insertion in the Notch1 locus and in tumors generated in the TAL1/, TAL1/LMO2, OLIG2/LMO1, ThPOK and Ikaros mouse models, which harbored activating mutations in NOTCH1. Our results demonstrate that gene expression profiling identifies common oncogenic pathways in T-cell tumors generated in mice, establishes common mechanisms of transformation for several T-ALL oncogenes and allows coupling of poorly characterized genes identified in proviral insertional sites with well characterized oncogenes and downstream molecular pathways. The identification of mechanisms of T-cell transformation common to tumors of different origin lays the ground for the identification of new therapeutic targets for the treatment of T-ALL.

The NOTCH1 signaling pathway plays a critical role in the regulation of hematopoietic stem cell homeostasis and is required for lymphocytes to adopt a T-cell fate. Importantly, aberrant activation of NOTCH signaling due to activating mutations in the NOTCH1 gene is involved in the pathogenesis of over 50% human T-cell acute lymphoblastic leukemias (T-ALL) and the therapeutic efficacy of inhibition of NOTCH signaling via gamma-secretase inhibitors (GSI) is currently being tested in clinical trials. However, little is known about the transcriptional programs activated downstream of NOTCH1 activation that contribute to the transformation of T-cell progenitors. Here we used chromatin immunoprecipitation and promoter microarrays (ChIP-on-chip) to identify direct transcriptional targets of NOTCH1; and microarray gene expression analysis to decipher the oncogenic transcriptional network activated by the NOTCH1 oncoprotein in T-ALL cells. Using the Hu19K arrays that contain over 13,000 human promoter regions, we have identified 134 candidate direct targets (p Inhibition of NOTCH1 signaling with compound E, a strong GSI, induced a gene expression signature characterized by the downregulation of known NOTCH1 direct target genes such as DELTEX1, HES1 and HEY1. Analysis of different cell lines representative of different stages of maturation arrest and different oncogenic groups of T-ALL identified a core signature of transcriptional responses to NOTCH signaling inhibition, which included the downregulation of the MYC oncogene as well as numerous genes involved in nucleotide metabolism and protein synthesis. In addition, cell cycle inhibitors p27/KIP1 and p18/INK4D were upregulated. Importantly, 13 of the top direct target genes of NOTCH1 identified by ChIP-on-chip were consistently downregulated (p

Risk stratification remains limited for patients being treated for T-ALL due to a lack of biologic predictors of outcome. As a consequence, treatment assignment on modern protocols has been largely achieved through random assignment. Recent observations have suggested that overexpression of specific gene(s) may provide a reliable means to risk stratify patients. We hypothesized that microarray analysis may identify gene sets that distinguish both therapeutic response and patient outcome in T-ALL. We analyzed the gene expression profiles of 45 primary T-ALL samples (24 CCR, 21 relapse) from a matched, case control study with sufficient cRNA for microarray analysis (COG #8704). We performed oligonucleotide microarray analysis using Affymetrix U133Av.2 genechips which have approximately 54,000 target genes and ESTs. Following heirarchical clustering in dChip and R Language analyses (Chiaretti et al. Blood, 2004), but using RMA normalization, we identified 37 genes that serve as reliable predictors of CCR or relapse. Leave-one-out least discriminant analysis cross-over validation further constrained our prognostic gene identifiers to 21 genes of robust significance. These 21 genes predict 87 % of CCR and 82% of relapse accurately (p

The Ikaros and Notch1 genes are critical to T-cell formation and differentiation through transcriptional activation of target genes and interaction with chromatin remodeling complexes. Notch1 is a cell surface receptor that is cleaved upon activation to enter the nucleus, and mutated in over 50% of human T-ALL. Ikaros is a zinc finger transcription factor that acts as a homodimer or heterodimer paired with one of several Ikaros family related proteins. Dominant negative Ikaros isoforms generated by alternative splicing activities can induce T-cell tumors in mice and have been reported to participate in the pathogenesis of human T-cell leukemia. An ENU-induced point mutant allele of Ikaros named Plstc which inhibits activity of the normal isoform as well as of other Ikaros family members has been shown to cause a hereditary T-cell lymphoma with autosomal dominant transmission in C57B6 mice. We characterized this disease in heterozygote carriers (Plstc+/−). We now demonstrate for the first time that Notch1 activating mutations are present at a high frequency in the T-cell malignancies that arise in Plstc +/− mice. More than half of Plstc +/− mice developed cervical lymph node enlargement by 4 months of age, with disease-associated mortality close to 100% by 8 months. Pathologic examination revealed massive involvement of the thymus, spleen, liver and kidneys. Peripheral blood smears demonstrated a leukemic phase, but no significant differences in hemoglobin levels or platelet counts. FACS analysis of cervical lymph node tumors revealed a population of lymphocytes overwhelmingly CD3+/T-cell receptorβ+ and minimal CD34, CD117 or T-cell receptorγδ expression. 17% to 63% of T-cells in the samples were CD4+/CD8+, a characteristic of incompletely differentiated thymic lymphocytes, and 36% to 54% of T-cells in samples expressed CD25. Symptomatic lymphoma was preceded by expansion of a CD4+/CD8+ T cell population in the peripheral blood by 10 weeks of age and as early as 8 weeks. This is earlier than reported for other dominant negative forms of Ikaros which do not alter activity of Ikaros related family members. Mutation analysis of Notch1 in Plstc+/− T-cell lymphomas revealed the presence of activating mutations in Notch1 in 9 of 15 cases (60%), analogous to those reported in human T-ALL samples. In these cases, the isolated lymph node predominantly contained either an exon 26 point mutation within the activating cleavage domain, or an exon 34 insertion leading to a frameshift and early truncation of the C-terminal PEST domain. By contrast, DNA isolated from lymph nodes of wild type littermates and DNA isolated from tail tips of each mouse by 5 weeks of age did not contain Notch1 mutations consistent with a somatically acquired oncogenic event. The predominance of Notch1 mutations in the tumors of Plstc+/− mice shows that preferential interactions between Notch1 and Ikaros family members can be a significant early event in T-cell proliferation and tumorigenesis, and provides a mouse model for further defining interactions between the Ikaros and Notch1 oncoproteins in the pathogenesis of T-cell tumors.

The Philadelphia translocation, encoding the BCR-ABL1 (BCR-ABL) fusion gene, is typically found in chronic myeloid leukemia (CML) and precursor B-cell acute lymphoblastic leukemia (B-ALL), but is exceptionally rare in T-cell acute lymphoblastic leukemia (T-ALL). To study the potential involvement of ABL1 gene rearrangements in T-cell malignancies, we screened 90 T-ALL cases by fluorescence in situ hybridization (FISH), using BCR and ABL1 probes. No BCR-ABL1 fusion signals were observed, confirming the low frequency of this rearrangement in T-ALL, but we did observe marked amplification (> 10 signals per nucleus) ABL1 of in 5 of 90 (5.5 %) T-ALL patients. Amplification of ABL1 occurred on small extrachromosomal elements that were not detectable by conventional cytogenetics. and hence are referred to as episomes. FISH, and array-CGH analyses delineated the amplicon as a 500 kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (CAN). Molecular analysis led to the identification of a NUP214-ABL1 fusion gene, which is generated as result of the circularization of the genomic region between ABL1 and NUP214 to form the episomes. This is the first example of an oncogenic fusion gene generated by extrachromosomal amplification. The NUP214-ABL1 transcript was detected in 5 patients with ABL1 amplification, in 5 of 85 (5.8 %) additional T-ALL patients, and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib mesylate (STI-571). The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased expression TLX1 of (HOX11) or TLX3 (HOX11L2), and with deletion of CDKN2A (p16), consistent with a multi-step pathogenesis of T-ALL. Our results identify a novel mechanism for the generation of a fusion gene on extrachromosomal elements, and indicate the importance of activated tyrosine kinase signaling in the pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of T-ALL patients that could benefit from imatinib treatment.

NOTCH1 was discovered originally through its involvement in a rare (7;9) translocation found in human T cell acute lymphoblastic leukemia (T-ALL). Here, we report that >50% of human T-ALLs have activating NOTCH1 mutations, occurring as amino acid substitutions in an extracellular heterodimerization (HD) domain and/or as frameshift and stop codon mutations that result in the deletion of a C-terminal PEST destruction box. Normal pro-NOTCH1 is processed into a heterodimer consisting of an extracellular subunit and a transmembrane subunit, which associate non-covalently through the HD domain. NOTCH1 activation is triggered by binding of Serrate or Delta-like ligands to the extracellular subunit, which induces successive proteolytic cleavages in the transmembrane subunit that are dependent on i) metalloproteases and ii) gamma-secretase. The γ-secretase cleavage releases intracellular NOTCH1 (ICN1), which translocates to the nucleus and forms a transcriptional activation complex with the transcription factor CSL and co-activators of the Mastermind family. Normal turnover of ICN1 is regulated by the C-terminal PEST sequence. Data pointing to the existence of frequent abnormalities of NOTCH1 in T-ALL stemmed from a functional screen of 30 T-ALL cell lines. This identified five T-ALL cell lines that underwent growth arrest in response to i) treatment with an inhibitor γ-secretase, and ii) retroviral transduction of dominant negative Mastermind-like-1. Sequencing of of cDNAs from 4 of these 5 cell lines demonstrated both a missense mutation in the HD domain and a frameshift mutation in the PEST domain lying in cis in the same NOTCH1 allele. Subsequent sequencing of genomic DNA obtained from bone marrow lymphoblasts of 96 children and adolescents with T-ALL demonstrated identical or similar mutations in NOTCH1 in 53 samples (55.2%). Mutations in the HD domain alone were observed in 26 cases (27.1%), in the PEST domain alone in 11 cases (11.4%), and in both the HD and PEST domains in 16 cases (16.7%). Mutations were observed in tumors associated with expression of HOX11 (2/3), HOX11L2 (10/13; 77%), TAL1 (12/31; 39%), LYL1 (9/14; 64%), MLL-ENL (1/3) or CALM-AF10 (1/2), which span the major molecular T-ALL subtypes. In contrast, NOTCH1 mutations were not observed in genomic DNAs samples obtained from B-ALL lymphoblasts (N=89), or from T-ALL patients with NOTCH1-associated disease at the time of clinical remission (N=4). Reporter gene assays conducted with plasmids expressing normal and mutated forms of NOTCH1 showed that a PEST deletion or various HD mutations alone caused ~1.5-fold and 3–9-fold stimulations of reporter gene activity, respectively, whereas normal NOTCH1 lacked intrinsic signaling activity. More strikingly, the combination of various HD mutations and a PEST deletion in cis caused synergistic 20–40-fold stimulations of reporter gene activity that were completely abrogated by a γ-secretase inhibitor, indicating that signaling depends on proteolysis. These results suggest a model in which HD domain mutations promote ICN1 production, and PEST domain mutations enhance ICN1 stability. Our findings greatly expand the role of NOTCH1 in the pathogenesis of human T-ALL, and provide a rationale for targeted therapies that interfere with NOTCH signaling.

TAL1 is a basic helix-loop-helix (bHLH) protein that is essential for hematopoiesis. Activation of the TAL1/SCL gene occurs in as many as 60% of patients with pediatric and adult T cell acute lymphoblastic leukemia (T-ALL). Mouse models have demonstrated that TAL1 transforms by inhibiting the function of E proteins, however the transcriptional targets of TAL1 that contribute to leukemia are not known. In this study we sought to identify targets of TAL1 that contribute to the transformation of thymocytes through siRNA-mediated knockdown of TAL1 expression in the Jurkat T-ALL cell line that expresses high levels of TAL1. We have generated several clonal cell lines that stably express a siRNA directed against TAL1 with up to 90% knockdown of TAL1 at both the RNA and protein level. Phenotypic characterization of these cells revealed that Jurkat cells with decreased TAL1 expression display a decreased proliferative capacity. This phenotype is augmented in lower serum concentrations in which wild-type and siRNA control Jurkat cells proliferate rapidly. Microarray expression analysis using Affymetrix U133 Plus 2.0 arrays has revealed that TAL1 is a global regulator of gene expression and that many pathways are affected by TAL1 knockdown, including those implicated in cell growth and proliferation. This data will enable us to dissect the downstream pathways of TAL1 by knocking-down or overexpressing these TAL1 targets (depending on whether they are up- or down-regulated in the absence of TAL1), and assessing whether they affect the proliferative rate or survival T-ALL cells. We anticipate that we will identify and validate potential therapeutic targets for TAL1-induced leukemia using this combination of approaches.

Aberrant expression of transcription factor oncogenes is a common feature in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), however, oncogenic transcriptional programs downstream of T-ALL oncogenes are mostly unknown. We used chromatin immunoprecipitation combined with promoter microarrays (ChIP on chip) to identify direct transcriptional targets of TAL1/SCL, a bHLH transcription factor aberrantly expressed in 60% of patients with T-ALL. Using the Hu12K arrays that contain over 12,000 human promoter regions, we have identified 71 direct targets of TAL1 in the Jurkat T-ALL cell line by ChIP on chip, and have been able to validate 80% of them by conventional chromatin immunoprecipitation followed by quantitative real time PCR. It has previously been demonstrated that TAL1 needs to bind to E proteins (E2A, HEB or E2-2) to achieve an efficient interaction with DNA. We verified that promoters occupied by TAL1 are bound by both the E-proteins E2A and HEB in at least 50% of TAL1-bound promoter regions, suggesting that TAL1/E2A as well as TAL1/HEB heterodimers play a role in the transformation of T-cell precursors. Using RNA interference to knock down TAL1, we have demonstrated functional regulation through TAL1-binding of a significant percentage of the targets identified by ChIP on chip. Results demonstrate that some TAL1 direct targets are repressed by the binding of this transcription factor, while others seem to be activated or are not altered, indicating a high level of complexity in the organization of TAL1-mediated transcriptional regulatory networks. Also, specific association of the expression levels of TAL1 targets in TAL1 positive primary leukemia samples has been shown by comparing gene expression arrays from the different T-ALL subsets. Our results indicate that TAL1 acts as a master transcriptional regulator at the top of a complex regulatory network that contributes to leukemogenesis by regulating T-cell differentiation, proliferation and survival.

Here, we have investigated the role of the Notch pathway in the generation and maintenance of KrasG12V-driven non-small cell lung carcinomas (NSCLCs). We demonstrate by genetic means that γ-secretase and RBPJ are essential in the formation of NSCLCs. Importantly, pharmacologic treatment of mice carrying autochthonous NSCLCs with a γ-secretase inhibitor (GSI) blocks cancer growth. Treated carcinomas present reduced HES1 levels and, interestingly, reduced phosphorylated ERK without changes in phosphorylated MEK. Mechanistically, we show that HES1 directly binds and represses the promoter of DUSP1, encoding a dual phosphatase active against phospho-ERK. Accordingly, GSI treatment upregulates DUSP1 and decreases phospho-ERK. These data provide proof for the in vivo therapeutic potential of γ-secretase inhibitors in primary NSCLCs.

The D-type cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. Cyclin D3 gene is rearranged and the protein is overexpressed in several human lymphoid malignancies. In order to determine the function of cyclin D3 in development and oncogenesis, we generated and analyzed cyclin D3-deficient mice. We found that cyclin D3−/− animals fail to undergo normal expansion of immature T lymphocytes and show greatly reduced susceptibility to T cell malignancies triggered by specific oncogenic pathways. The requirement for cyclin D3 also operates in human malignancies, as knock-down of cyclin D3 inhibited proliferation of acute lymphoblastic leukemias deriving from immature T lymphocytes. These studies point to cyclin D3 as a potential target for therapeutic intervention in specific human malignancies.

Oncogenic NOTCH signaling is a major driver of T-cell transformation in T-cell acute lymphoblastic leukemia (T-ALL). However, clinical studies testing the efficacy of NOTCH1 inactivation with γ-secretase inhibitors (GSIs) have shown limited antileukemic activity for these drugs as single agents. Here we used an expression-based virtual screening approach and network perturbation analyses to identify and functionally characterize new highly active antileukemic drugs synergistic with NOTCH1 inhibition in T-ALL. Gene expression profiling studies have shown a prominent gene expression signature dominated by genes involved in growth and metabolism downstream of NOTCH1 in T-ALL. Notably, loss of the PTEN tumor suppressor gene confers resistance to GSI therapy and effectively rescues the gene expression signature induced by NOTCH1 inhibition in T-ALL. We hypothesized that drugs inducing transcriptional programs overlapping with those driven by NOTCH1 inhibition and antagonizing those resulting from PTEN loss could have synergistic antileukemic effects with GSIs in PTEN wild type and PTEN null leukemia cells. To address this question we generated gene expression signatures from Pten conditional-inducible knockout NOTCH1-driven leukemias in basal condition, upon NOTCH1 inhibition by GSI treatment and upon deletion of Pten. Connectivity Map (cMAP) analysis in this series identified 17 high scoring compounds as candidate antileukemic drugs (p To address the mechanisms mediating the antileukemic effects of withaferin A we performed a detailed analysis of the gene expression signatures induced by this drug in T-ALL lymphoblasts. These studies revealed a strong enrichment of downregulated genes involved in translation regulation in T-ALL cells upon treatment with withaferin A (p Disclosures Califano: Therasis Inc: Employment; Cancer Genetics Inc: Consultancy; Ipsen pharmaceuticals: Consultancy; Thermo Fischer Scientific: Consultancy.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas frequently associated with poor prognosis and for which genetic mechanisms of transformation remain incompletely understood. Using RNA sequencing and targeted sequencing, here we identify a recurrent in-frame deletion (VAV1 Δ778-786) generated by a focal deletion-driven alternative splicing mechanism as well as novel VAV1 gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7) in PTCL. Mechanistically these genetic lesions result in increased activation of VAV1 catalytic-dependent (MAPK, JNK) and non-catalytic-dependent (nuclear factor of activated T cells, NFAT) VAV1 effector pathways. These results support a driver oncogenic role for VAV1 signaling in the pathogenesis of PTCL.

Abstract 851 Clinical studies have demonstrated that acute myeloid leukemia (AML) is heterogeneous with respect to presentation and to clinical outcome, and many studies have shown that cytogenetics can be used to improve prognostication. More recently, it has been shown that FLT3, NPM1, and CEBPA mutations can be used to further risk stratify AML patients. Although recent studies have identified novel, recurrent somatic mutations in AML in genes including TET2, ASXL1, IDH1, IDH2, and PHF6, their prognostic value has not yet been evaluated among patients treated on a large phase III clinical trial. Here we report full-length DNA resequencing of FLT3, NPM1, CEBPA, H/K/NRAS, KIT, WT1, TET2, ASXL1, IDH1/2, TP53, RUNX1, PTEN, and PHF6 in pre-treatment genomic DNA from 398 patients with de novo AML younger than 60 years of age enrolled in the ECOG E1900 protocol. We could identify a clonal alteration, defined as a somatic mutation or cytogenetic abnormality, in 91.2% of all patients; 42% had 1 somatic alteration, 36.4% had 2 alterations, 11.3% had 3 alterations and 1.5% had 4 or more somatic alterations. Mutational Analysis: We identified somatic mutations in FLT3 (37% total; 30% ITD, 7% TKD), NPM1 (14%), CEBPA (10%), TET2 (10%), NRAS (10%), WT1 (10%), KIT (9%), IDH2 (8%), IDH1 (6%), RUNX1 (6%), ASXL1 (4%), PHF6 (3%), KRAS (2.5%), TP53 (2%), and PTEN (1.5%) (Figure 1A). We then performed correlation analysis to determine the frequency of different mutational combinations in AML patients. In addition to previously identified mutational correlations (FLT3 and NPM1, KIT and core binding factor leukemia), we found that FLT3 and ASXL1 mutations were mutually exclusive in this large cohort (p =0.0008). In addition, we found that IDH1/IDH2 mutations were mutually exclusive of both TET2 (p=0.02), and WT1 (p=0.01) mutations consistent with overlapping roles in AML pathogenesis (Figure 1B). Integration of mutational data with response data and outcome: Mutations in ASXL1 (CR rate 33.3% versus 66.5% for ASXL1-WT patients; p= 0.009) were enriched in patients who did not respond to induction chemotherapy. We found that mutations in FLT3 (p=0.0005), ASXL1 (p=0.005; Figure 1C), and PHF6 (p=0.02) were associated with reduced OS. Notably, the adverse effects of ASXL1 and PHF6 mutations on outcome were restricted to patients treated with standard dose daunorubicin (p Figure 1. A) Circos diagram depicting relative frequency and pairwise co-occurrence of mutations in de novo AML patients enrolled in the ECOG protocol E1900. B) Circos diagram showing mutational events in IDH1/2, TET2, and WT1 mutant AML patient. C) Overall survival in AML patients with and without ASXL1 mutations (p=0.005). D) Overall survival in AML patients with and without IDH2 R172 and R140 mutations (p=0.003). Figure 1. A) Circos diagram depicting relative frequency and pairwise co-occurrence of mutations in de novo AML patients enrolled in the ECOG protocol E1900. B) Circos diagram showing mutational events in IDH1/2, TET2, and WT1 mutant AML patient. C) Overall survival in AML patients with and without ASXL1 mutations (p=0.005). D) Overall survival in AML patients with and without IDH2 R172 and R140 mutations (p=0.003). Disclosures: No relevant conflicts of interest to declare.

A cDNA encoding a novel member of the cysteine proteinase family of proteins has been cloned from a human breast carcinoma cDNA library, by using a polymerase chain reaction-based cloning strategy. The isolated cDNA contains an open reading frame coding for a polypeptide of 321 amino acids that has been tentatively called cathepsin O. This protein presents all the structural features characteristic of the different cysteine proteinases identified to date, including the active site cysteine residue that is involved in covalent intermediate formation during peptide hydrolysis. The cathepsin O cDNA was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade the synthetic peptides benzyloxycarbonyl-Phe-Arg-7-amido-4- methylcoumarin and benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin widely used as substrates for cysteine proteinases. Cathepsin O proteolytic activity was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), an inhibitor of this subclass of proteolytic enzymes, thus providing additional evidence that the isolated cDNA codes for an authentic cysteine proteinase. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that cathepsin O is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in normal cellular protein degradation and turnover.