ALBERT

Description: Stress-induced angiogenesis enormously contributes to both normal development and pathogenesis of various diseases including cancer. Among many stress response pathways implicated in regulation of angiogenesis, the amino acid response (AAR) and the unfolded protein response (UPR) pathways are closely interconnected, as they converge on the common target, eIF2α, which is a key regulator of protein translation. Two kinases, namely Gcn2 (Eif2ak4) and Perk (Eif2ak3), are responsible for transducing signals from AAR and UPR, respectively, to phosphorylation of eIF2α. Even though numerous studies have been performed, this close interconnection between AAR and UPR makes it difficult to clearly distinguish different contributions of these two pathways in regulation of angiogenesis. In this study, we generated a zebrafish angiogenic model harboring a loss-of-function mutation of the threonyl-tRNA synthetase (tars) gene. Tars belongs to a family of evolutionarily conserved enzymes, aminoacyl-tRNA synthetases (aaRSs), which control the first step of protein translation through coupling specific amino acids with their cognate tRNAs. Deficiencies of several aaRSs in zebrafish have been shown to cause increased branching of blood vessels, and this angiogenic phenotype has roughly been explained by activation of AAR and UPR; however, it is unclear whether both AAR and UPR are required and to what extent they contribute to this process. To address this issue, we first performed RNA-seq analyses of Tars-mutated and control zebrafish embryos, as well as those with knockdown of either Gcn2 or Perk in both genotypes. We found that the AAR target genes are dramatically activated in the Tars-mutants, whereas the genes associated with the three UPR sub-pathways (i.e., Perk-, Ire1- and Atf6-mediated pathways) remain inactive, except for very few genes (e.g., Atf3, Atf4, Asns and Igfbp1) that are shared in both AAR and UPR, thus suggesting activation of AAR, but not UPR, in the Tars-mutants. In support of this notion, knockdown of the AAR-associated kinase Gcn2 in the Tars-mutants largely represses the activated genes, while the Perk knockdown shows very little effect. Nonetheless, in contrast to the apparently dispensable role of Perk in Tars-mutants, knockdown of Perk in control embryos leads to specific gene expression alterations, suggesting that Perk effectively functions in homeostatic states (i.e., controls), but, in the stress condition (i.e., Tars-mutants), its function is largely overwhelmed by activation of the Gcn2-mediated AAR. To validate these observations, we investigated the angiogenic phenotypes of the zebrafish models upon genetic and pharmacological interference with the AAR and UPR pathways. A transgenic zebrafish line, Tg(flk1:EGFP), was crossed with the Tars-mutants to visualize angiogenesis in vivo. We observed increased branching of blood vessels in the Tars-mutants, which is rescued by tars mRNA but not an enzymatically dead version. Importantly, knockdown of Gcn2 in the Tars-mutants rescues this phenotype. In contrast, knockdown of Perk, or knockdown of two other known eIF2α kinases, Hri (Eif2ak1) or Pkr (Eif2ak2), shows no effect. Furthermore, knockdown of either one of two major factors downstream to eIF2α, namely Atf4 and Vegfα, or inhibition of Vegf receptor with the drug SU5416, also rescue the phenotype. Thus, these results confirm that AAR, but not UPR, is required for the Tars-deficiency-induced angiogenesis. Taken together, this study demonstrates that, despite being closely interconnected and even sharing a common downstream target, the Gcn2-mediated AAR and the Perk-mediated UPR can be activated independently in different conditions and differentially regulate cellular functions such as angiogenesis. This notion reflects the specificity and efficiency of multiple stress response pathways that are evolved integrally to benefit the organism by ensuring sensing and responding precisely to different types of stresses. This study also provides an example of combining systematic gene expression profiling and phenotypic validations to distinguish activities of such interconnected pathways. Further clarification of the mechanisms shall advance our understanding of how the organisms respond to diverse stresses and how the abnormalities in these regulatory machineries cause cellular stress-related diseases such as cancer, diabetes and immune disorders. Disclosures No relevant conflicts of interest to declare.

Description: Acquisition of additional genetic and/or epigenetic abnormalities other than BCR/ABL fusion gene is believed to cause disease progression in chronic myeloid leukemia (CML) from chronic phase to blast phase. To gain insights into the underlying mechanisms, we screened DNA samples from CML patients during acute transformation for alterations in a number of transcription factor genes crucial to myeloid-lymphoid development. In 85 cases of CML blast transformation, we identified two new mutations in the coding region of GATA-2, a negative regulator of hematopoietic stem/progenitor cell differentiation. L359V within zinc finger domain (ZF) 2 of GATA-2 was found in 8 cases with myelo-monoblastic features, while an in-frame deletion of six amino acids (D341–346) across the border of ZF1 was detected in 1 patient at blast crisis with eosinophilia. Further studies showed that L359V not only increased transactivation activity, but also enhanced inhibitory effects on the major myelopoietic regulator PU.1. Consistent with the myelo-monoblastic features of CML patients with GATA-2 L359V mutant, transduction of GATA-2 L359V mutant into HL-60 cells or BCR/ABL-harboring mouse model disturbed myelo-monocytic differentiation/proliferation in vitro and in vivo, respectively. These data suggest that GATA-2 mutations may be involved in acute myeloid transformation in some CML patients.

Description: Abstract 2489 Purpose: Standard Induction chemotherapy (Ara-C/daunorubicin, 3+7 regimen) in elderly patients (pts) with AML results in approximately 35–45% complete remission (CR) rate, and pts with resistant disease (RD) have a median survival of only 1–3 months. Developing a test for accurate prediction of response to standard induction therapy at the time of diagnosis may help inform treatment selection and improve clinical trial design. Methods: Single cell network profiling (SCNP) is a multi-parameter flow cytometry based approach for simultaneous interrogation of intracellular signaling pathways at a single cell level. SCNP was used to evaluate signaling profiles in leukemic blasts and to develop a classifier (DXSCNP) of response to induction therapy (CR/CRi, i=incomplete) in a Training Set of cryopreserved diagnostic samples (57 PB and 43 BM) collected from 74 non-M3 AML pts, aged 56+ treated with 3+7-based regimens on 4 SWOG clinical trials. SCNP intracellular readouts quantifying apoptotic response after 24 hrs in vitro treatment with Ara-C/Daunorubicin formed the inputs for DXSCNP. Pt and disease characteristics available either at diagnosis, i.e. relevant to induction therapy choices (e.g., age, WBC counts, FAB class, secondary AML, performance status – CLINICAL1), or available after start of induction therapy (CLINICAL2) were used to develop 2 clinical predictors (DXCLINICAL1 and DXCLINICAL2) of CR/CRi in the Training Set. The performance characteristics of these 3 classifiers were then tested separately in BM and PB sample sets. Specifically, classifier validation was performed in 2 independent BM sample sets (A: n=24 BM samples from ECOG E3999 trial; B: n=42 independent BM samples from the same 4 SWOG trials from which the Training samples were derived) and in 1 PB sample set (C: n=53, from the 4 SWOG trials; notably only 24 patients were shared between Set B and C)). The area under the receiver operating characteristic curve (AUROC) was used to measure each classifier's ability to predict response to 3+7 induction therapy. Out of bag estimates (OOB) of AUROC were calculated using the Training Set, and H0:AUROC=0.5 was tested against HA:AUROC〉0.5 for each classifier in the Validation Sets. Results: As shown in Table 1, DXSCNP was a significant predictor of CR/CRi in BM samples. The AUROC for the DXSCNP classifier was 0.81 in the Training Set and 0.76 (p=0.01) and 0.72 (p=0.02) in Validation Sets A and B, respectively. No significant DXCLINICAL1 was identified from the 74 pts in the Training Set(OOB AUROC∼0.5). By contrast the AUROC of DXCLINICAL2 (which included inputs for cytogenetics, Flt3ITD, and NPM1 mutational status) was 0.63 (OOB) in the Training Set, and 0.61 (p=0.18) and 0.53 (p=0.38) in Validation Sets A and B, respectively. Of note, DXSCNP remained significant (p=0.03 and 0.04) when controlling for DXCLINICAL2 in both sample sets. Similar performance of DXSCNPwas observed in different pre-specified subgroups (although of small sample sizes) defined by pt, sample or disease characteristics. Using PB samples, the AUROCs for the DXSCNP classifier were, 0.87 (OOB) in the Training Set and 0.53 (p=ns) in the Validation Set C. However, the performance of DXSCNP in PB samples differed significantly between secondary vs. De novo AML i.e. AUROC= 0.24 vs 0.8 respectively. Of note, DXSCNPperformed similarly for the subset of de novo patients for whom paired BM with PB samples were tested (AUC=0.78 (PB) and 0.74 (BM)). Conclusion: This is the first study describing the successful validation of an SCNP-based classifier to predict response to standard induction chemotherapy in elderly AML pts with performance superior to clinical variables available at diagnosis. A similar SCNP classifier with overlapping cellular biology inputs was previously validated for pediatric AML. These results confirm the ability of quantitative SCNP readouts to provide independent and actionable information on disease biology and pt treatment choices. Independent validation in prospective studies is warranted. Disclosures: Cesano: Nodality, Inc: Employment, Equity Ownership. Gayko:Nodality, Inc.: Employment, Equity Ownership. Putta:Nodality, Inc.: Employment, Equity Ownership. Louie:Nodality, Inc.: Employment, Equity Ownership. Westfall:Nodality, Inc.: Employment, Equity Ownership. Purvis:Nodality, Inc.: Employment, Equity Ownership. Spellmeyer:Nodality, Inc.: Employment, Equity Ownership. Marimpietri:Nodality, Inc.: Employment, Equity Ownership. Hackett:Nodality, Inc.: Employment, Equity Ownership. Shi:Nodality, Inc.: Employment, Equity Ownership.

Description: Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2187 Poster Board II-164 Chronic myeloid leukemia (CML) progression is characterized by occurrence of new cytogenetic and molecular abnormalities. In the previous study, we have shown the important role of GATA-2 L359V mutation in CML progression. To further ascertain the truth of transcription factor GATA-2 in hematological malignancies, we expanded our study to GATA-2 full length by directly sequencing and applied MassARRAY assay into GATA-2 L359V mutation analysis. Finally, no GATA-2 L359V mutation was found in 270 acute myeloid leukemia, 30 myelodysplastic syndrome, 50 acute lymphoblastic leukemia, 12 chronic lymphocytic leukemia, 40 CML chronic phase and 286 BCR/ABL negative myeloproliferative disorders except CML blast crisis. A new variation of GATA-2 resulted in P250A change was identified, which was not found to have statistical difference between patients with hematological malignancies and healthy control. Hence, we concluded GATA-2 L359V is exclusively associated with CML progression but not other hematological malignancies and P250A is a new single nucleotide polymorphism. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 180 Background and Aims. The main pathogenic molecular events associated with myeloproliferative neoplasms (Polycythemia Vera, Essential Thrombocytosis, and Primary Myelofibrosis) are mutations in Janus kinase 2 (JAK2) or in the thrombopoietin receptor that arise in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. The approved JAK2 inhibitor (Ruxolitinib) is not expected to be selective for the mutant JAK2/receptor signaling or to completely suppress the multiple signaling pathways activated by the aberrant JAK2 signaling. We postulate that myeloproliferative neoplasms can be treated more effectively if we target the constitutive JAK2 signaling by a JAK2 inhibitor together with another kinase inhibitor targeting a specific pathway that is co-activated by the aberrant JAK2 signaling. This should increase targeting specificity, reduce JAK2 inhibitor dosages, and minimize potential side effects of these drugs. To this end, we constructed cell line models of myeloproliferative neoplasms and tested the models using a JAK2 inhibitor in combination with a panel of kinase inhibitors to identify combination pairs that give the best synergism. The synergistic pair was further confirmed in mouse models of myeloproliferative neoplasms. Methods. Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-Abl. The effect of two JAK2 inhibitors (Ruxolitinib and TG101348) in combination with a panel of 15 various kinase inhibitors (one JNK, one B-Raf, one ROCK-1, one TIE-2, one PI3K, two CDK, two MAPK, three p38, and three mTOR inhibitors). An 8×8 constant ratio Latin square design were used for testing inhibition of cell proliferation/survival in these cell line models. Calculations were carried out using the Chou-Talalay method to determine which drug-pair demonstrated synergism in inhibiting cell growth. Further eight PI3K inhibitors were acquired and tested when we found strong synergism between the JAK2 inhibitors and the PI3K inhibitor ZSTK474 in the first panel. The engineered Ba/F3 cells were also inoculated into female BALB/c nude mice to generate the JAK2 mutant mouse model. These mice were treated intravenously with Ruxolitinib and the PI3K inhibitor GDC0941. Blood profile and physical parameters of the mice were measured for 14 days post treatment. Bone marrow cells from mice reconstituted with bone marrow from JAK2 V617F knock-in mice were plated for colony formation in the presence or absence of Ruxolitinib and the PI3K inhibitor GDC0941. Primary Epo-independent colonies from CD34+ cells of one PV patient were assessed in two independent experiments in the presence or absence of combination drugs. Results. Out of 15 kinase inhibitors tested, three PI3K inhibitors (ZSTK474, GDC0941 and BEZ235), synergized with JAK2 inhibitors (Ruxolitinib and TG101348) in inhibiting cell growth. The combination index was less than 0.5 in all 8×8 dose combination ratios. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as growth of Ba/F3 cells expressing Bcr-Abl (at equivalent STAT5 activation levels) was unaffected by this combination treatment. Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight due to proliferation of autonomous cells. Our combination treatment using Ruxolitinib and GDC0941 could drastically reduce spleen weight compared to treatment with either compound alone. Endogenous erythroid colony forming unit (CFU-E) and burst forming unit (BFU-E) formation from JAK2 V617F knock-in bone marrow cells was reduced significantly by the combined use of Ruxolitinib and GDC0941 compared to individual drugs. Similarly, Epo-independent BFU-E colony formation from peripheral CD34+ cells of one JAK2 V617F-positive PV patient was reduced significantly by the drug combination. Conclusions. Our findings of strong synergy between the JAK2 inhibitors and PI3K inhibitors suggested that we may be able to administer these drugs at lower concentrations than when the drugs are used individually. It provides a framework for combination trials using compounds in these two classes in patients with myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

Description: Natural-killer/T cell lymphoma (NKTCL) is a malignant proliferation of CD56+/cytoCD3+ lymphocytes and constitutes a heterogeneous group of aggressive lymphoma prevalent in Asian and South American populations. NKTCL represents a distinct clinicopathologic entity of non-Hodgkin’s lymphoma, characterized by male predominance, strong association with Epstein-Barr virus (EBV) infection, prominent tissue necrosis and aggressive clinical course. However, molecular pathogenesis of NKTCL remains largely elusive. Here we identified somatic mutations by whole-exome sequencing in 25 NKTCL patients and extended validation through targeted sequencing in an additional 80 cases. Functional experiments including RNA unwinding test, colony forming assay, cell proliferation assay and gene expression profiling were also performed. Overall, 50.5% of NKTCL patients displayed somatic mutations of RNA helicase family, tumor suppressors (TP53 and MGA), and/or epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). Recurrent mutations were most frequently discovered in RNA helicase gene DDX3X (21/105 cases, 20.0%). Mutations of DDX3X were seldom overlapped with those of TP53. Functionally, DDX3X mutants exhibited reduced RNA unwinding activity and enhanced cell proliferation. Similar stimulatory effect on cell proliferation was observed in cells transfected with specific siRNA targeting DDX3X. Gene expression profiling revealed an association of DDX3X mutations with activation of NF-kB and MAPK pathways. The clinical follow-up data showed that DDX3X-mutated patients presented a poor prognosis. Our work suggests the heterogeneity of gene mutational spectrum of NKTCL and provides a potential therapeutic target for relevant cases. Disclosures No relevant conflicts of interest to declare.

Description: Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease with distinct biology and clinical features. According to clinical and biology process, CML can be divided as two different phase: the initial chronic phase (CP) and progression phase including accelerated phase (AP) followed by blast crisis (BC). In a previous study, we have identified the transcription factor GATA-2 L359V mutation in CML BC patients but not CP and its pivotal role in CML progression (PNAS 2008 105:2076–2081). Here, we continued to explore the occurrence of GATA-2 L359V mutation in other hematological malignancies using Mass-ARRAY assay and sequence analysis. A total of 652 patient samples were included in our study. These patients were referred to 270 Acute Myeloid Leukemia (AML) including M1–M7, 30 Myelodysplastic Syndrome (MDS), 50 Acute Lymphoblastic Leukemia (ALL), 12 Chronic Lymphocytic Leukemia (CLL), 40 CML CP and 250 BCR/ABL negative Myeloproliferative Disorder (MPD) patients. 5 ml bone marrow sample before therapy was collected with informed consent and genomic DNA was isolated. The diagnosis of AML, ALL, CML, CLL, MDS and MPD was established according to the 2001 WHO diagnostic criteria. In addition, 8 samples of CML BC harboring GATA-2 L359V were supplied into our study as positive control and 100 peripheral blood samples of healthy adult as normal control. As a result, no L359V mutation was detected in AML, ALL, MDS, CLL, BCR/ABL negative MPD and CML CP. Our data strongly suggested that GATA-2 L359V is solely associated with CML progression but not other hematological malignancies. Therefore, we favored this idea, i.e., BCR/ABL underlies the pathogenic basis of CML CP; However, subsequent genomic instabilities contribute to mutation of key transcription factor such as GATA-2 which ultimately trigger the blastic transformation of CML.

Description: To evaluate the prognostic value of genetic mutations for acute myeloid leukemia (AML) patients, we examined the gene status for both fusion products such as AML1 (CBFα)–ETO, CBFβ-MYH11, PML-RARα, and MLL rearrangement as a result of chromosomal translocations and mutations in genes including FLT3, C-KIT, N-RAS, NPM1, CEBPA, WT1, ASXL1, DNMT3A, MLL, IDH1, IDH2, and TET2 in 1185 AML patients. Clinical analysis was mainly carried out among 605 cases without recognizable karyotype abnormalities except for 11q23. Of these 605 patients, 452 (74.7%) were found to have at least 1 mutation, and the relationship of gene mutations with clinical outcome was investigated. We revealed a correlation pattern among NPM1, DNMT3A, FLT3, IDH1, IDH2, CEBPA, and TET2 mutations. Multivariate analysis identified DNMT3A and MLL mutations as independent factors predicting inferior overall survival (OS) and event-free survival (EFS), whereas biallelic CEBPA mutations or NPM1 mutations without DNMT3A mutations conferred a better OS and EFS in both the whole group and among younger patients 〈 60 years of age. The use of molecular markers allowed us to subdivide the series of 605 patients into distinct prognostic groups with potential clinical relevance.

Description: The positive regulatory domain I (PRDM1) is a master regulator in the differentiation of mature B lymphocytes to plasma cells. It has 2 isoforms, PRDM1α and PRDM1β, and is regulated by the transcriptional regulator nuclear factor kappa (NF)–κB. PRDM1 protein expression was recently demonstrated in a subset of diffuse large B-cell lymphoma (DLBCL) with aggressive behavior, a type of lymphoma for which rituximab associated with chemotherapy (R-CHOP) is now widely indicated. Using laser microdissection combined with reverse transcription–polymerase chain reaction (RT-PCR) amplification, PRDM1 gene expression was assessed in 82 DLBCL patients. The results showed that both PRDM1α and PRDM1β transcripts were expressed in microdissected lymphoma cells only in the non–germinal center B-cell–like (non-GCB) subtype of DLBCL. PRDM1β gene expression was correlated with short survival time in the non-GCB patients treated with CHOP but not with R-CHOP. In vitro, B-lymphoma cells resistant to chemotherapy expressed PRDM1β. Rituximab suppressed PRDM1β expression, which was concomitant with NF-κB inactivation. The value of PRDM1β expression as a prognostic marker in non-GCB DLBCL might thus be considered. This study confirms the efficiency of rituximab on DLBCL and allows a better understanding of one of its biologic actions.