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The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å cocrystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology.

Somatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5′-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy–induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome., National Institute for Health Research (Great Britain). Biomedical Research Centre, Hope Funds for Cancer Research Grillo-Marxuach Family Fellow

The Notch signaling pathway is a regulator of self-renewal and differentiation in several tissues and cell types. Notch is a binary cell-fate determinant, and its hyperactivation has been implicated as oncogenic in several cancers including breast cancer and T-cell acute lymphoblastic leukemia (T-ALL). Recently, several studies also unraveled tumor-suppressor roles for Notch signaling in different tissues, including tissues where it was before recognized as an oncogene in specific lineages. Whereas involvement of Notch as an oncogene in several lymphoid malignancies (T-ALL, B-chronic lymphocytic leukemia, splenic marginal zone lymphoma) is well characterized, there is growing evidence involving Notch signaling as a tumor suppressor in myeloid malignancies. It therefore appears that Notch signaling pathway’s oncogenic or tumor-suppressor abilities are highly context dependent. In this review, we summarize and discuss latest advances in the understanding of this dual role in hematopoiesis and the possible consequences for the treatment of hematologic malignancies.

Background Protein phosphatase 2A (PP2A) represents a family of potent tumor suppressors that are often suppressed in human cancers by upregulation of proteins that inhibit subunit assembly into active enzyme complexes. Thus, restoration of PP2A has assumed increasing importance for cancer treatment. In earlier work, we found that perphenazine (PPZ) kills leukemic T cells by activating PP2A (Gutierrez et al. 2014. J Clin Invest.). PPZ also acts to inhibit dopamine D2 receptor (DRD2) in the basal ganglia, which causes movement disorders at dosages less than those needed to kill T-ALL cells, effectively precluding "repurposing" of PPZ for the therapy of T-ALL. Hypothesis We sought to identify PPZ analogues that activate PP2A and induce apoptosis in T-ALL cells but lack the ability to bind and inhibit DRD2. Such analogues would not induce the movement disorders that have limited the usefulness of PPZ as an anti-leukemic drug. Methods PP2A is a heterotrimeric phosphatase that is assembled from three classes of subunits, encoded by two possible "A" genes, 15 "B" genes and 2 "C" genes, providing 60 distinct PP2A holoenzymes that can potentially form in the cell. To identify the key subunits of PP2A required for the antitumor activity of PPZ in T-ALL cells, we knocked out each subunit in human T-ALL cell lines and treated these cells with PPZ. Biochemical reporter assays were established to identify a phenothiazine derivative, iHAP1, that efficiently reactivates PP2A but does not inhibit dopamine signaling. iHAP1 preclinical studies included in vivo efficacy and toxicity in T-ALL xenograft models and in vitro efficacy in 248 cancer cell lines from 35 distinct tumor types. Results Using this approach, we found that knockout of each of three specific subunit genes of PP2A - PPP2R1A, PPP2CA and PPP2R5E - uniquely conferred resistance to PPZ treatment in T-ALL cell lines,. An independent Immunoprecipitation followed by western blotting indicated that all three subunits form a functional PP2A heterotrimeric holoenzyme complex in response to PPZ treatment. Narla and coworkers have published extensively about a series of compounds called "small molecule PP2 activators" or SMAPs (Sangodkar et al. 2017 J Clin Invest.). Intriguingly, we showed that activities of SMAP compounds depend on a different "B subunit" - PPP2R2A - and target different signal transduction pathways. Based on this finding, we sought to identify analogues of PPZ that more potently activate PP2A through this mechanism and kill T-ALL cells but lack inhibitory activity against DRD2. Testing more than 80 analogues of PPZ revealed a highly potent PP2A activator, iHAP1 (improvedHeterocyclic Activators of PP2A 1). iHAP1 is ten times more potent than PPZ in its ability to activate PP2A and kill tumor cells, but does not measurably inhibit dopamine signalling. iHAP1 is highly active as an antitumor drug human T-ALL xenograft models, without causing untoward movement disorders or other toxicity in vivo. Phosphoproteomics analysis followed by detailed biochemical assays revealed that the potent antitumor activity of PPZ and iHAP1 is mediated by dephosphorylation of MYBL2, a transcription factor that is essential for expression of genes whose products mediate prometaphase, and thus for cancer cell growth and survival. SMAP compounds do not dephosphorylate the same transcription factor and rather target other phosphoproteins. Thus, the potent PP2A activator iHAP1 drives three specific PP2A subunits into an active trimeric phosphatase and this drug is highly active against T-ALL and other hematologic malignancies. iHAP1 did not exhibit measurable toxicity up to 80 mg/kg/day PO or IM for 30 days in preclinical studies of human leukemia xenografts growing in immunosuppressed mice, providing a therapeutic index for this lead compound in preclinical studies. Conclusions Our findings show that small molecules promote the assembly of unique PP2A complexes with different regulatory subunits and substrates, allowing detailed structure and function studies of PP2A family members. A goal is to identify small molecules that assemble PP2A enzymes containing each of the remaining 13 regulatory PP2A subunits, thereby targeting a diverse array of substrates crucial to the pathogenesis of cancer and other diseases. Disclosures Gray: Gatekeeper: Equity Ownership; Syros: Equity Ownership; Petra: Equity Ownership; C4: Equity Ownership; B2S: Equity Ownership; Soltego: Equity Ownership; Novartis: Research Funding; Takeda: Research Funding; Astellas: Research Funding; Taiho: Research Funding; Janssen: Research Funding; Kinogen: Research Funding; Voronoi: Research Funding; Her2llc: Research Funding; Deerfield: Research Funding; Sanofi: Research Funding. Fischer:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Deerfield: Consultancy, Research Funding; Astellas: Research Funding.

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18hi1727/+ embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.

Introduction: Pediatric and young adult patients (pts) with relapsed/refractory (R/R) acute leukemia have a poor prognosis. Selinexor is a first-in-class antagonist of exportin (XPO1) with single agent activity in adults with acute myelogenous leukemia (AML). Dose-defining non-hematologic toxicities in adult studies included nausea, weight loss and anorexia. Methods: DFCI Protocol 13-563 was a Phase I dose-escalation trial conducted at nine institutions to test the safety, tolerability and pharmacokinetic (PK) characteristics of selinexor administered to pediatric and young adult pts with R/R acute leukemias. Selinexor was orally administered twice weekly during 4-week cycles. Three dose levels (DLs) were tested (30 mg/m2, 40 mg/m2 and 56 mg/m2) with dose-escalation decisions based on standard 3+3 Phase I design rules. Required supportive care medications included antiemetics and bacterial prophylaxis. All grade ≥2 non-hematologic adverse events (AEs) were recorded with the exception of non-clinically significant grade 2 laboratory abnormalities. Grades 1-4 hematologic AEs were expected in a relapsed leukemia patient population and were not considered AEs. Results: Sixteen pts enrolled on study, including 5 with primary refractory disease and 7 with second or greater relapse; 6 pts had received prior hematopoietic stem cell transplant. Median age was 9.5 years (range 2.3-21.8). Twelve pts had AML and 4 had acute lymphoblastic leukemia (ALL). Median number of completed cycles was 1 (range 0-3). Thirteen pts (81%) were evaluable for dose limiting toxicity (DLT) assessment, having received at least 7 doses of selinexor during the first cycle. Of 9 pts enrolled at DL1 (30 mg/m2), 6 were DLT-evaluable; 1 pt experienced a DLT of grade 5 pancreatitis (possibly related to study treatment). Three pts enrolled at DL1 were not DLT-evaluable; 2 pts were removed from study due to disease progression and 1 pt died from complications of a pulmonary infection. Six pts enrolled at DL2 (40 mg/m2) and none experienced a DLT. One pt was treated at DL3 (56 mg/m2) and developed a DLT of cognitive impairment (grade 3). Further enrollment onto DL3 was suspended secondary to an amendment capping dose escalation at 40 mg/m2 (equivalent to recommended single-agent dose in adult AML). During Cycle 1, the most common selinexor treatment-related AE's were hyponatremia (31%), elevated alanine aminotransferase (25%), nausea (25%), hyperkalemia (25%), elevated aspartate aminotransferase (19%) and diarrhea (19%). The most common grade ≥ 3 selinexor treatment-related AE's were hyponatremia (31%), elevated alanine aminotransferase (19%), hyperglycemia (13%) and hyperkalemia (13%). In contrast to adults treated with selinexor, there were no cases of grade ≥ 3 weight loss or nausea and only one case of grade 3 anorexia. One AML pt, after 2 cycles, achieved a complete remission with incomplete recovery of platelet count and one ALL pt, after 1 cycle, achieved a partial response, resulting in a protocol-defined overall response rate of 12.5%. Two of 4 pts with ALL (one each in DLs 1 and 2) developed clinically significant (grade 4) TLS. These included the pt who achieved a PR and another pt with early T-cell precursor ALL. Overall, 9 pts (56%), including 6 AML and 3 ALL, experienced an objective response and/or an investigator-defined clinical benefit (e.g., reduction in transfusions, clearance of peripheral blasts, decreased pain) from therapy. Conclusions: Selinexor, at a doses of 30 or 40 mg/m2 given twice weekly, was well tolerated in pediatric and young adult pts with R/R acute leukemia, demonstrating single agent clinical activity and less GI toxicity than had been seen in adults. The high frequency of TLS in ALL pts is particularly notable as treatment of ALL with selinexor has not been previously reported. Analysis of pharmacokinetic and correlative biology studies are underway. Based on the results of this Phase I study, the recommended Phase II dose of single-agent selinexor in children with R/R acute leukemia is 40 mg/m2 administered twice weekly. These results also support the continued investigation of selinexor in combination with multiagent chemotherapy. Disclosures Burke: AMGEN: Speakers Bureau; JAZZ: Speakers Bureau; Shire: Speakers Bureau.

LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid–rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL.

Comprehensive analysis of the cancer genome has become a standard approach to identifying new disease loci, and ultimately will guide therapeutic decisions. A key technology in this effort, single nucleotide polymorphism arrays, has been applied in hematologic malignancies to detect deletions, amplifications, and loss of heterozygosity (LOH) at high resolution. An inherent challenge of such studies lies in correctly distinguishing somatically acquired, cancer-specific lesions from patient-specific inherited copy number variations or segments of homozygosity. Failure to include appropriate normal DNA reference samples for each patient in retrospective or prospective studies makes it difficult to identify small somatic deletions not evident by standard cytogenetic analysis. In addition, the lack of proper controls can also lead to vastly overestimated frequencies of LOH without accompanying loss of DNA copies, so-called copy-neutral LOH. Here we use examples from patients with myeloid malignancies to demonstrate the superiority of matched tumor and normal DNA samples (paired studies) over multiple unpaired samples with respect to reducing false discovery rates in high-resolution single nucleotide polymorphism array analysis. Comparisons between matched tumor and normal samples will continue to be critical as the field moves from high resolution array analysis to deep sequencing to detect abnormalities in the cancer genome.

To identify dysregulated pathways in distinct phases of NOTCH1-mediated T-cell leukemogenesis, as well as small-molecule inhibitors that could synergize with or substitute for γ-secretase inhibitors (GSIs) in T-cell acute lymphoblastic leukemia (T-ALL) therapy, we compared gene expression profiles in a Notch1-induced mouse model of T-ALL with those in human T-ALL. The overall patterns of NOTCH1-mediated gene expression in human and mouse T-ALLs were remarkably similar, as defined early in transformation in the mouse by the regulation of MYC and its target genes and activation of nuclear factor-κB and PI3K/AKT pathways. Later events in murine Notch1-mediated leukemogenesis included down-regulation of genes encoding tumor suppressors and negative cell cycle regulators. Gene set enrichment analysis and connectivity map algorithm predicted that small-molecule inhibitors, including heat-shock protein 90, histone deacetylase, PI3K/AKT, and proteasome inhibitors, could reverse the gene expression changes induced by NOTCH1. When tested in vitro, histone deacetylase, PI3K and proteasome inhibitors synergized with GSI in suppressing T-ALL cell growth in GSI-sensitive cells. Interestingly, alvespimycin, a potent inhibitor of the heat-shock protein 90 molecular chaperone, markedly inhibited the growth of both GSI-sensitive and -resistant T-ALL cells, suggesting that its loss disrupts signal transduction pathways crucial for the growth and survival of T-ALL cells.

Activation of β-catenin has been causatively linked to the etiology of colon cancer. Conditional stabilization of this molecule in pro-T cells promotes thymocyte development without the requirement for pre-TCR signaling. We show here that activated β-catenin stalls the developmental transition from the double-positive (DP) to the single-positive (SP) thymocyte stage and predisposes DP thymocytes to transformation. β-Catenin–induced thymic lymphomas have a leukemic arrest at the early DP stage. Lymphomagenesis requires Rag activity, which peaks at this developmental stage, as well as additional secondary genetic events. A consistent secondary event is the transcriptional up-regulation of c-Myc, whose activity is required for transformation because its conditional ablation abrogates lymphomagenesis. In contrast, the expression of Notch receptors as well as targets is reduced in DP thymocytes with stabilized β-catenin and remains low in the lymphomas, indicating that Notch activation is not required or selected for in β-catenin–induced lymphomas. Thus, β-catenin activation may provide a mechanism for the induction of T-cell–acute lymphoblastic leukemia (T-ALL) that does not depend on Notch activation.

In this report, inhibitors of the gamma-secretase enzyme have been exploited to characterize the antiproliferative relationship between target inhibition and cellular responses in Notch-dependent human T cell acute lymphoblastic leukemia (T-ALL) cell lines. Inhibition of gamma-secretase led to decreased Notch signaling, measured by endogenous NOTCH intracellular domain (NICD) formation, and was associated with decreased cell viability. Flow cytometry revealed that decreased cell viability resulted from a G(0)/G(1) cell cycle block, which correlated strongly to the induction of apoptosis. These effects associated with inhibitor treatment were rescued by exogenous expression of NICD and were not mirrored when a markedly less active enantiomer was used, demonstrating the gamma-secretase dependency and specificity of these responses. Together, these data strengthen the rationale for using gamma-secretase inhibitors therapeutically and suggest that programmed cell death may contribute to reduction of tumor burden in the clinic.

Aberrant expression of 1 or more transcription factor oncogenes is a critical component of the molecular pathogenesis of human T-cell acute lymphoblastic leukemia (T-ALL); however, oncogenic transcriptional programs downstream of T-ALL oncogenes are mostly unknown. TAL1/SCL is a basic helix-loop-helix (bHLH) transcription factor oncogene aberrantly expressed in 60% of human T-ALLs. We used chromatin immunoprecipitation (ChIP) on chip to identify 71 direct transcriptional targets of TAL1/SCL. Promoters occupied by TAL1 were also frequently bound by the class I bHLH proteins E2A and HEB, suggesting that TAL1/E2A as well as TAL1/HEB heterodimers play a role in transformation of T-cell precursors. Using RNA interference, we demonstrated that TAL1 is required for the maintenance of the leukemic phenotype in Jurkat cells and showed that TAL1 binding can be associated with either repression or activation of genes whose promoters occupied by TAL1, E2A, and HEB. In addition, oligonucleotide microarray analysis of RNA from 47 primary T-ALL samples showed specific expression signatures involving TAL1 targets in TAL1-expressing compared with -nonexpressing human T-ALLs. Our results indicate that TAL1 may act as a bifunctional transcriptional regulator (activator and repressor) at the top of a complex regulatory network that disrupts normal T-cell homeostasis and contributes to leukemogenesis.

Gain and/or loss of function mediated by chimeric transcription factors generated by nonrandom translocations in leukemia is a key to understanding oncogenesis. E2A-hepatic leukemia factor (HLF), a chimeric basic region/leucine zipper (bZIP) transcription factor expressed in t(17;19)-positive leukemia cells, contributes to leukemogenesis through its potential to inhibit apoptosis. To identify physiologic counterparts of this chimera, we investigated the function of other bZIP factors that bind to the same DNA sequence recognized by E2A-HLF. Here, we show that thyrotroph embryonic factor (TEF), which shares a high level of sequence identity with HLF and recognizes the same DNA sequence, is expressed in a small fraction of each subset of hematolymphoid progenitors. When TEF was introduced into FL5.12 interleukin 3 (IL-3)-dependent cells, TEF protected the cells from apoptosis due to IL-3 deprivation. Unexpectedly, TEF also almost completely down-regulated expression of the common beta (betac) chain of cytokine receptors. Consequently, TEF-expressing cells accumulated in G(0)/G(1) phase without undergoing apoptosis. These findings suggest that TEF is one of the apoptotic regulators in hematopoietic progenitors and controls hematopoietic-cell proliferation by regulating the expression of the betac chain. In contrast, E2A-HLF promoted cell survival more efficiently than TEF but did not down-regulate betac chain expression, suggesting that E2A-HLF retains ideal properties for driving leukemic transformation.

PU.1 is a member of the Ets family of transcription factors and plays an essential role in the development of both myeloid and lymphoid cells. To examine zebrafish pu.1 (zpu.1) expression in subpopulations of blood cells during zebrafish development, we linked a 9-kb zebrafish genomic fragment upstream of the zpu.1 initiator codon to green fluorescent protein (GFP) and microinjected this construct to generate stable transgenic lines. GFP-positive fluorescent myeloid precursors were observed migrating from the anterolateral mesoderm in living embryos from 16 to 28 hours after fertilization (hpf) in a pattern that overlaps the expression pattern of endogenous zpu.1 mRNA. Analysis of larval histologic sections revealed GFP-expressing hematopoietic cells in the developing zebrafish kidney. Flow cytometric analysis of cells from adult whole kidney marrow revealed 2 discrete subpopulations of GFP-positive cells, which after cell sorting exhibited either myeloid or early lymphoid morphology. Thus, the zebrafish zpu.1 promoter fragment used here is capable of driving reporter gene expression in subsets of embryonic and adult hematopoietic cells. These transgenic lines will be useful to dissect the cellular and molecular control of myeloid cell differentiation, and this promoter fragment may prove useful in the development of zebrafish models of acute myeloid leukemia.

Rearrangements of the MLL locus, located on human chromosome 11q23, are frequent in both infant and therapy-related leukemias. Gene expression analysis of MLL-rearranged B-precursor acute lymphoblastic leukemias (MLL B-ALLs) has identified these cases as a unique subtype of leukemia, characterized by the expression of genes associated with both lymphoid and myeloid hematopoietic lineages. Here we show that MLL fusions also generate a distinct genetic subtype of T-lineage ALL (MLL T-ALL), in which leukemic cells are characterized by an early arrest in thymocyte differentiation, with suggestive evidence of commitment to the γδ lineage. Interestingly, multiple genes linked to cell proliferation (eg, PCNA, MYC, CDK2, and POLA) were down-regulated in MLL-fusion samples, relative to those transformed by other T-ALL oncogenes (P < .000 001, Fisher exact test). Overall, MLL T-ALL cases consistently demonstrated increased levels of expression of a subset of major HOX genes—HOXA9, HOXA10, and HOXC6—and the MEIS1 HOX coregulator (P < .008, one-sided Wilcoxon test), a pattern of gene expression that was reiterated in MLL B-ALLs. However, expression of myeloid lineage genes, previously reported in MLL B-ALLs, was not identified in T-lineage cases with this abnormality, suggesting that myeloid gene dysregulation is dispensable in leukemic transformation mediated by MLL fusion proteins. Our findings implicate dysregulation of HOX gene family members as a dominant mechanism of leukemic transformation induced by chimeric MLL oncogenes. (Blood. 2003;102:262-268)

Genome-wide chemical mutagenesis screens in the zebrafish(Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafishmpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, andL-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis.

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation, is thought to drive the leukemic transformation of early B-cell precursors by repressing an evolutionarily conserved apoptotic pathway. To test this hypothesis, we sought to identify downstream targets of E2A-HLF in t(17;19)+ pro-B leukemia cells (UOC-B1) that had been transfected with a zinc-inducible vector encoding a dominant-negative suppressor (E2A-HLF[dn]) of the oncoprotein. Representational difference analysis of mRNAs from E2A-HLF(dn)+ UOC-B1 cells grown with (E2A-HLF inactive) or without (E2A-HLF active) the addition of zinc yielded several differentially expressed cDNA fragments that were individually subcloned. Two of the clones, designated F-5 and G-4, hybridized with mRNAs that were upregulated by E2A-HLF. Levels of both transcripts declined sharply within 8 to 12 hours after suppression of E2A-HLF DNA-binding activity, becoming undetectable after 96 hours. The F-5 cDNA was identified as a portion of ANNEXIN VIII, whose product was expressed in promyelocytic leukemia cells and UOC-B1 cells, but not in other leukemic cell lines. A novel full-length cDNA cloned with the G-4 fragment encoded a protein that we have named SRPUL (sushi-repeat protein upregulated in leukemia). It is normally expressed in heart, ovary, and placenta, but could not be detected in leukemic cell lines other than UOC-B1. Neither protein prevented apoptosis in interleukin-3–dependent murine pro-B cells, suggesting that they have paraneoplastic roles in leukemias that express E2A-HLF, perhaps in the disseminated intravascular coagulopathy and hypercalcemia that characterize these cases.

Leukemic lymphoblasts expressing the E2A-HLF oncoprotein possess wild-type p53 genes, but do not undergo apoptosis in response to DNA damage. Experimentally, E2A-HLF prevents apoptosis due to growth factor deprivation or γ-irradiation in interleukin-3 (IL-3)–dependent murine pro-B cells. To directly test the chimeric protein’s ability to abrogate p53-mediated cell death, we used mouse myeloid leukemia cells (M1p53tsval) that constitutively express a temperature-sensitive (ts) mutant p53 gene and undergo apoptosis when p53 assumes an active wild-type configuration. This effect is blocked by treatment with IL-6, which allows the cells to survive in culture despite wild-type p53 activation. We introduced E2A-HLF into M1p53tsval cells and found that they were resistant to p53-mediated apoptosis and that E2A-HLF effectively substituted for the survival functions of IL-6. The expression of p53-responsive genes such as p21 and Bax was upregulated normally, suggesting that E2A-HLF acts downstream of p53 to block execution of the p53-induced apoptotic program. NFIL3, a growth factor-regulated bZIP protein that binds to the same DNA-consensus site as E2A-HLF, delays apoptosis in IL-3–dependent pro-B cells deprived of growth factor. By contrast, in the present study, enforced expression of NFIL3 failed to protect M1p53tsval cells from p53-dependent apoptosis and actively antagonized the ability of IL-6 to rescue cells from that fate, consistent with its role as either a transcriptional repressor or activator, depending on the cell type in which it is expressed. We conclude that the E2A-HLF chimera abrogates p53-induced apoptosis in leukemic cells, possibly through the transcriptional modulation of cell death pathways that are activated by p53 in response to DNA damage. © 1998 by The American Society of Hematology.

Leukemic lymphoblasts expressing the E2A-HLF oncoprotein possess wild-type p53 genes, but do not undergo apoptosis in response to DNA damage. Experimentally, E2A-HLF prevents apoptosis due to growth factor deprivation or γ-irradiation in interleukin-3 (IL-3)–dependent murine pro-B cells. To directly test the chimeric protein’s ability to abrogate p53-mediated cell death, we used mouse myeloid leukemia cells (M1p53tsval) that constitutively express a temperature-sensitive (ts) mutant p53 gene and undergo apoptosis when p53 assumes an active wild-type configuration. This effect is blocked by treatment with IL-6, which allows the cells to survive in culture despite wild-type p53 activation. We introduced E2A-HLF into M1p53tsval cells and found that they were resistant to p53-mediated apoptosis and that E2A-HLF effectively substituted for the survival functions of IL-6. The expression of p53-responsive genes such as p21 and Bax was upregulated normally, suggesting that E2A-HLF acts downstream of p53 to block execution of the p53-induced apoptotic program. NFIL3, a growth factor-regulated bZIP protein that binds to the same DNA-consensus site as E2A-HLF, delays apoptosis in IL-3–dependent pro-B cells deprived of growth factor. By contrast, in the present study, enforced expression of NFIL3 failed to protect M1p53tsval cells from p53-dependent apoptosis and actively antagonized the ability of IL-6 to rescue cells from that fate, consistent with its role as either a transcriptional repressor or activator, depending on the cell type in which it is expressed. We conclude that the E2A-HLF chimera abrogates p53-induced apoptosis in leukemic cells, possibly through the transcriptional modulation of cell death pathways that are activated by p53 in response to DNA damage. © 1998 by The American Society of Hematology.

Although prognosis has improved for children with T-cell acute lymphoblastic leukemia (T-ALL), 20% to 30% of patients undergo induction failure (IF) or relapse. Leukemia-initiating cells (LICs) are hypothesized to be resistant to chemotherapy and to mediate relapse. We and others have shown that Notch1 directly regulates c-Myc, a known regulator of quiescence in stem and progenitor populations, leading us to examine whether c-Myc inhibition results in efficient targeting of T-ALL–initiating cells. We demonstrate that c-Myc suppression by small hairpin RNA or pharmacologic approaches prevents leukemia initiation in mice by eliminating LIC activity. Consistent with its anti-LIC activity in mice, treatment with the BET bromodomain BRD4 inhibitor JQ1 reduces C-MYC expression and inhibits the growth of relapsed and IF pediatric T-ALL samples in vitro. These findings demonstrate a critical role for c-Myc in LIC maintenance and provide evidence that MYC inhibition may be an effective therapy for relapsed/IF T-ALL patients.

Loss of the chromatin remodeling ATPase CHD5 has been linked to the progression of neuroblastoma tumors, yet the underlying mechanisms behind the tumor suppressor role of CHD5 are unknown. In this study, we purified the human CHD5 complex and found that CHD5 is a component of the full NuRD transcriptional repressor complex, which also contains methyl-CpG binding proteins and histone deacetylases. The CHD5/NuRD complex appears mutually exclusive with the related CHD4/NuRD complex as overexpression of CHD5 results in loss of the CHD4 protein in cells. Following a search for genes that are regulated by CHD5 in neuroblastoma cells, we found that CHD5 binds to and represses the G2/M checkpoint gene WEE1. Reintroduction of CHD5 into neuroblastoma cells represses WEE1 expression, demonstrating that CHD5 can function as a repressor in cells. A catalytically inactive mutant version of CHD5 is able to associate with a NuRD cofactor but fails to repress transcription. Our study shows that CHD5 is a NuRD-associated transcriptional repressor and identifies WEE1 as one of the CHD5-regulated genes that may link CHD5 to tumor suppression.

Approximately 5% to 10% of all non-Hodgkin's lymphomas contain a t(2; 5)(p23; q35) chromosomal rearrangement, which we have previously shown results in the generation of the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). To assess the transforming potential of NPM-ALK in an animal model, we infected 5-fluorouracil–treated murine bone marrow using retroviral stocks and transplanted this infected marrow into lethally irradiated BALB/cByJ mice. Male mice were transplanted with bone marrow from female donors at 10 weeks of age, with 7 of the animals receiving marrow infected with a retroviral construct, pSRαMSVtkneo-NPM-ALK, that contains the human NPM-ALK cDNA, and 4 serving as a control group, receiving “empty” pSRαMSVtkneo-infected marrow. Whereas all mice in the control group were alive and well up to 11 months after transplantation, 4 of the 7 mice transplanted with marrow containing the NPM-ALK construct developed lymphoma within 4 to 6 months. Tumors arose in the mesenteric lymph nodes, with metastases to the lungs, kidneys, liver, spleen, and the paraspinal area. When cells from the tumors and bone marrow were transplanted into sublethally irradiated secondary recipients, 10 of these 13 mice developed tumors within 9 months. Immunoblot analysis of cell lysates using an ALK polyclonal antibody showed NPM-ALK expression in all tumors examined. Histologically, the tumors were composed of a uniform population of large immunoblastic cells with basophilic cytoplasm, centrally placed nuclei, and distinct nucleoli. Genotypic analysis showed that the tumors were B-lineage and clonal, with rearrangements of the Ig heavy- and κ light-chain loci and no rearrangements of the T-cell receptor β locus. Immunocytochemical studies confirmed the presence of IgM heavy chains and κ light chains within the tumor cells. Thus, in this retroviral gene transfer model, NPM-ALK expression in mice causes B-lineage large-cell lymphoma, suggesting a direct causative role for this activated fusion tyrosine kinase in human lymphoma.

This report describes the prognostic significance of the intensity of surface membrane antigen expression in a series of 1,231 children older than 1 year with newly diagnosed B-precursor acute lymphoblastic leukemia (ALL) treated on Pediatric Oncology Group (POG) treatment protocols. All patients had dual-color flow cytometric immunophenotyping performed at a central reference laboratory with a standard panel of monoclonal antibodies. The flow cytometers used in the study were calibrated with a standard fluorescence microparticle that permitted conversion of relative fluorescence channels to standard units of mean equivalents of soluble fluorochrome (MESF). In univariate analysis, fluorescence intensity of CD45 and CD20 was significantly associated with event-free survival (EFS), whereas other markers showed no significant correlation with outcome. Patients whose blasts were greater than the 75th percentile of intensity for CD45 (corresponding to 18,000 MESF units with CD45-FITC, or about 8% of the intensity of normal lymphocytes) fared significantly worse than those with lower-density CD45, and those whose blasts were greater than the 25th percentile of intensity for CD20 (corresponding to 17,900 MESF units with CD20-PE) had a poorer EFS. The intensity of both CD45 and CD20 was independently correlated with outcome. There was no significant correlation between intensity of expression of either antigen and traditional clinical risk factors, ploidy, or t(9; 22) or t(1; 19). All patients with t(4; 11) had CD45 intensity greater than the 75th percentile, but CD45 intensity retained its prognostic significance after adjusting for t(4; 11). In multivariate analysis, both CD45 intensity greater than the 75th percentile and CD20 intensity greater than the 25th percentile were significantly correlated with poor outcome independently of previously reported poor prognostic factors including National Cancer Institute (NCI) risk group, ploidy, trisomies of 4 and 10, and adverse translocations including t(1; 19), t(9; 22), and t(4; 11). We conclude that in childhood B-precursor ALL, the intensity of expression of CD20 and CD45 provides prognostic information not available from simple consideration of antigen expression as positive or negative, and adds to that obtained from traditional clinical and biologic risk factors.

BMX is a member of the TEC family of nonreceptor tyrosine kinases. We have used structure-based drug design in conjunction with kinome profiling to develop a potent, selective, and irreversible BMX kinase inhibitor, BMX-IN-1, which covalently modifies Cys496. BMX-IN-1 inhibits the proliferation of Tel-BMX-transformed Ba/F3 cells at two digit nanomolar concentrations but requires single digit micromolar concentrations to inhibit the proliferation of prostate cancer cell lines. Using a combinatorial kinase inhibitor screening strategy, we discovered that the allosteric Akt inhibitor, MK2206, is able to potentiate BMX inhibitor's antiproliferation efficacy against prostate cancer cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the transformation of T-cell progenitors. Although the prognosis of this disease has substantially improved due to the introduction of intensified chemotherapy, the clinical outcome of T-ALL patients with primary resistant or relapsed disease remains poor, indicating that further therapeutic improvement is urgently needed. We have previously demonstrated that activation of tyrosine kinase 2 (TYK2) contributes to aberrant survival of human T-ALL cells. TYK2 is a member of the Janus-activated kinase (JAK) tyrosine kinase family and our report was the first to implicate TYK2 in T-ALL pathogenesis. Indeed, our gene knockdown experiments showed TYK2 dependency in 14 of 16 (88%) T-ALL cell lines and 5 of 8 (63%) patient-derived T-ALL cells tested, suggesting that inhibition of TYK2 would be beneficial in most patients with T-ALL. Based on these findings, we investigated the therapeutic potential of a novel small-molecule TYK2 kinase inhibitor NDI-031301 in T-ALL. We found that NDI-031301 shows potent and selective inhibitory activity against TYK2 in a cellular context, because this compound strongly inhibited the growth of TYK2-transfomed Ba/F3 cells when compared to the JAK inhibitors tofacitinib and baricitinib, whereas Ba/F3 cells transformed by other tyrosine kinases showed decreased sensitivity to NDI-031301. NDI-031301 induced robust growth inhibition in each of 4 human T-ALL cell lines representing different molecular subtypes of the disease (DU.528, KOPT-K1, HPB-ALL and SKW-3), with IC50 values of 0.8186 - 2.380 μM after 72 hours of exposure. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with tofacitinib and baricitinib. To elucidate the mechanism of apoptosis induced by NDI-031301 in T-ALL cells, we next investigated cellular signaling pathways that are associated with cell survival and specifically affected by TYK2 inhibition with NDI-031301. Western blotting analysis demonstrated that treatment with 3 μM of NDI-031301 resulted in reduction of STAT1 Tyr-701 phosphorylation and BCL2 levels in KOPT-K1 cells, consistent with our previous finding that TYK2 phosphorylates STAT1 and upregulates BCL2 expression in most T-ALL cells. Surprisingly, the treatment also uniquely led to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage, which was not observed with tofacitinib and baricitinib. NDI-031301-mediated activation of SAPK/JNK and p38 MAPK pathways are likely mediated through inhibition of TYK2, because increased phosphorylation levels of SAPK/JNK and p38 MAPK were observed in the cells transfected with TYK2-targeting shRNAs, while the levels of ERK1/2 phosphorylation were not upregulated. Further investigation revealed that activation of p38 MAPK occurred within 1 hour of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacologic inhibition of p38 MAPK by SB203580 partially rescued apoptosis induced by TYK2 inhibitor, while inhibition of ERK or SAPK/JNK showed no rescue effects. Finally, we found that daily oral administration of NDI-031301 at 100mg/kg BID to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumor burden and a significant survival benefit. After 29 days of treatment, the mice receiving NDI-031301 had marked reductions in infiltration of leukemia cells into spleen and bone marrow by comparison with controls. Thus, our findings clearly support TYK2 inhibition with NDI-031301 or a related compound as a potential therapeutic strategy for patients with T-ALL, and also raise the possibility that enhancing p38 MAPK activation in T-ALL cells may be an approach to accentuate its anti-leukemic activity. Disclosures Masse: Nimbus Therapeutics: Employment. Miao:Nimbus Therapeutics: Employment. Rocnik:Nimbus Therapeutics: Employment. Kapeller:Nimbus Therapeutics: Employment.

LMO2is a crucial regulator of normal hematopoiesis but is progressively silenced from the early T-cell progenitor stage of thymic development. Aberrant LMO2 expression leads to a block in differentiation, increased self-renewal and aggressive T-cell acute lymphoblastic leukemia (T-ALL). Although ≈50% of T-ALL patients overexpress LMO2, this can be attributed to a cytogenetic lesion in only ≈10% of patients, leaving a significant portion mechanistically unaccounted for. We recently discovered somatic mutations that create an oncogenic super-enhancer driving TAL1 expression (Mansour et al., 2014, Science). Therefore, we investigated whether comparable mutations may be causing dysregulated LMO2 expression. Aberrant H3K27ac marks indicative of active chromatin were identified prior to and encompassing the non-coding exon 2 of the LMO2 gene by ChIP-seq in DU.528 and PF-382 T-ALL cell lines, both of which exhibit upregulated LMO2 expression but lack chromosomal lesions at this locus. Sequencing across this peak revealed a heterozygous 20bp duplication in PF-382 cells and a heterozygous 1bp deletion in DU.528 cells, located close to a region recently described as an intermediate promoter. Both indels generated a de novo MYB consensus motif. MYB ChIP-seq showed that ≥96% of reads aligned to the mutant rather than the wild-type allele, suggesting that MYB was preferentially recruited to the mutant allele. Heterozygous mutations were also detected in diagnostic samples from 3% (5/159) of pediatric and 6% (10/164) of adult T-ALL patients. Absence of the mutations in 7 available patient-matched remission samples confirmed that they were somatic. The mutations were densely distributed around highly conserved native ETS1, MYB and GATA motifs; 5 patients had an additional MYB site, 3 both a MYB and an ETS1 site, 1 an ETS1 site alone, and 4 had new RUNX1 binding sites. This suggests that this region may have a regulatory role that is exploited by the mutations to constitutively activate LMO2. All 6 mutant-positive patients with available RNA showed LMO2 overexpression as determined by qRT-PCR, consistent with the hypothesis that these mutations activate gene expression. Furthermore, monoallelic LMO2 expression could be demonstrated in DU.528 cells and 3 of 4 informative T-ALL samples using a heterozygous germline SNP. Expression of the mutations in luciferase reporter assays also indicated that they all markedly activated luciferase activity to between 2x and 57x compared to the wild-type sequence. To assess causality between the mutations and LMO2 dysregulation, we used CRISPR/Cas9 genome-editing with a guide RNA designed to target the mutant MYB site in PF-382 cells. Reduction of LMO2 expression to ≤10% of PF-382 mutant activity was observed in a clone where the mutant allele had been reverted to wild-type and in another clone with a single T>C substitution disrupting the mutant MYB binding site. Interestingly, 2 clones that increased the distance between the native and the mutant MYB sites also resulted in a reduction in LMO2 expression to 19% and 25% of PF-382 activity, suggesting that there are functionally limiting spatial constraints on the mutant MYB site in relation to other neighboring transcription factor binding sites. This was further validated by the lack of reduction in LMO2 expression in a clone where the sequence between the two MYB sites was altered but the spacing distance was unchanged. In conclusion, we have identified and functionally validated a novel recurrent mutation hotspot occurring in a non-coding site whereby introduction of additional binding sites for a number of different transcription factors drives monoallelic LMO2 overexpression from a neomorphic promoter in a substantial proportion of both adult and pediatric T-ALL patients. This mechanism of oncogene activation may be relevant to a wide variety of human cancers. Disclosures Fielding: Baxalta: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Ribosomal protein (RP) mutations in diseases such as 5q− syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ∼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.

Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34+ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

The BCL11B transcription factor is required for normal T-cell development, and has recently been implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) induced by TLX overexpression or Atm deficiency. To comprehensively assess the contribution of BCL11B inactivation to human T-ALL, we performed DNA copy number and sequencing analyses of T-ALL diagnostic specimens, revealing monoallelic BCL11B deletions or missense mutations in 9% (n = 10 of 117) of cases. Structural homology modeling revealed that several of the BCL11B mutations disrupted the structure of zinc finger domains required for this transcription factor to bind DNA. BCL11B haploinsufficiency occurred across each of the major molecular subtypes of T-ALL, including early T-cell precursor, HOXA-positive, LEF1-inactivated, and TAL1-positive subtypes, which have differentiation arrest at diverse stages of thymocyte development. Our findings provide compelling evidence that BCL11B is a haploinsufficient tumor suppressor that collaborates with all major T-ALL oncogenic lesions in human thymocyte transformation.

A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3′ untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP+ cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.

Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted, but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here, we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell–autonomous manner. Unexpectedly, loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly, null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation, thereby accelerating hematopoietic regeneration. Consistent with these findings, Puma is required for radiation-induced apoptosis in HSCs and HPCs, and Puma is selectively induced by irradiation in primitive hematopoietic cells, and this induction is impaired in Puma-heterozygous cells. Together, our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy.

To further unravel the molecular pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), we performed high-resolution array comparative genomic hybridization on diagnostic specimens from 47 children with T-ALL and identified monoallelic or biallelic LEF1 microdeletions in 11% (5 of 47) of these primary samples. An additional 7% (3 of 44) of the cases harbored nonsynonymous sequence alterations of LEF1, 2 of which produced premature stop codons. Gene expression microarrays showed increased expression of MYC and MYC targets in cases with LEF1 inactivation, as well as differentiation arrest at an early cortical stage of thymocyte development characterized by expression of CD1B, CD1E, and CD8, with absent CD34 expression. LEF1 inactivation was associated with a younger age at the time of T-ALL diagnosis, as well as activating NOTCH1 mutations, biallelic INK4a/ARF deletions, and PTEN loss-of-function mutations or activating mutations of PI3K or AKT genes. These cases generally lacked overexpression of the TAL1, HOX11, HOX11L2, or the HOXA cluster genes, which have been used to define separate molecular pathways leading to T-ALL. Our findings suggest that LEF1 inactivation is an important step in the molecular pathogenesis of T-ALL in a subset of young children.

Mutations in the human nucleophosmin (NPM1) gene are the most frequent genetic alteration in adult acute myeloid leukemias (AMLs) and result in aberrant cytoplasmic translocation of this nucleolar phosphoprotein (NPMc+). However, underlying mechanisms leading to leukemogenesis remain unknown. To address this issue, we took advantage of the zebrafish model organism, which expresses 2 genes orthologous to human NPM1, referred to as npm1a and npm1b. Both genes are ubiquitously expressed, and their knockdown produces a reduction in myeloid cell numbers that is specifically rescued by NPM1 expression. In zebrafish, wild-type human NPM1 is nucleolar while NPMc+ is cytoplasmic, as in human AML, and both interact with endogenous zebrafish Npm1a and Npm1b. Forced NPMc+ expression in zebrafish causes an increase in pu.1+ primitive early myeloid cells. A more marked perturbation of myelopoiesis occurs in p53m/m embryos expressing NPMc+, where mpx+ and csf1r+ cell numbers are also expanded. Importantly, NPMc+ expression results in increased numbers of definitive hematopoietic cells, including erythromyeloid progenitors in the posterior blood island and c-myb/cd41+ cells in the ventral wall of the aorta. These results are likely to be relevant to human NPMc+ AML, where the observed NPMc+ multilineage expression pattern implies transformation of a multipotent stem or progenitor cell.

To more comprehensively assess the pathogenic contribution of the PTEN-PI3K-AKT pathway to T-cell acute lymphoblastic leukemia (T-ALL), we examined diagnostic DNA samples from children with T-ALL using array comparative genomic hybridization and sequence analysis. Alterations of PTEN, PI3K, or AKT were identified in 47.7% of 44 cases. There was a striking clustering of PTEN mutations in exon 7 in 12 cases, all of which were predicted to truncate the C2 domain without disrupting the phosphatase domain of PTEN. Induction chemotherapy failed to induce remission in 3 of the 4 patients whose lymphoblasts harbored PTEN deletions at the time of diagnosis, compared with none of the 12 patients with mutations of PTEN exon 7 (P = .007), suggesting that PTEN deletion has more adverse therapeutic consequences than mutational disruptions that preserve the phosphatase domain. These findings add significant support to the rationale for the development of therapies targeting the PTEN-PI3K-AKT pathway in T-ALL.

Activating mutations in the NOTCH1 gene have been found in about 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL). In order to study the molecular mechanisms by which altered Notch signaling induces leukemia, a zebrafish model of NOTCH1-induced T-cell leukemia was generated using TAN-1, the NOTCH1 oncogene generated by the t(7;9)(q34;q34.3) chromosome translocation associated with human T-ALL. Seven of sixteen mosaic fish developed a T cell lymphoproliferative disease at about 5 months. These neoplastic cells extensively invaded tissues throughout the fish and caused an aggressive and lethal leukemia when transplanted into irradiated recipient fish. A stable transgenic fish line was then generated, which also develops leukemia, but with a longer latency for leukemia onset. This longer latency allowed crosses to be done to evaluate potential genetic interactions between NOTCH1 and other T-ALL oncogenes. Interestingly, LMO2 did not cooperate with NOTCH1 to induce T-ALL, while bcl2 had dramatic effects on latency and progression of T-ALL in this zebrafish model. These results suggest that the transforming functions of NOTCH1 and LMO2 may be redundant, while the functions of NOTCH1 and bcl2 are highly complementary. The ability of this model to detect a strong interaction between NOTCH1 and bcl2 suggests that genetic modifier screens have a high likelihood of revealing other genes that can cooperate with NOTCH1 to induce T-ALL.

Nucleophosmin (NPM1) is a multi-functional ubiquitous phosphoprotein that shuttles between the nucleolus and cytoplasm. Located on chromosome 5q35 NPM1 is involved in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) both as a chimeric fusion partner and as a putative key haploinsufficient tumor suppressor. Heterozygous NPMc+ mutations have been identified in 30% of AML, usually with normal karyotype. Such mutations create new nuclear export signals and disrupt the normal nucleolar localization signal, resulting in re-localization of both mutant and heterodimeric wild-type (WT) NPM protein from the nucleolus to the cytoplasm. The zebrafish is a model organism ideally suited to genotypic and phenotypic analysis of myelopoiesis and leukemogenesis, with a proven track record for facilitating the discovery of novel pathogenetic pathways. Using in silico analysis we identified two homologues (a common finding in zebrafish due to genome duplication during piscine evolution) of the human nucleophosmin gene in zebrafish. These two genes have been designated znpm1a and znpm1b. znpm1a is annotated by the National Center for Biotechnology (NCBI) while znpm1b is a known protein-coding region located via blast search of the human NPM1 amino acid sequence at www.ensembl.org/Danio_rerio/index.html. Whilst znpm1b exhibits slightly less identity to human NPM1 (47%) than znpm1a (64%) it demonstrates clear synteny with human chromosome 5q35 and mouse chromosome 11 (Figure 1). We confirmed expression of both znpm1a and znpm1b in embryonic tissue and adult hematopoietic tissue of the major lineages by RT-PCR of Green Fluorescent Protein (GFP)-sorted cells in pu.1-GFP transgenic zebrafish embryos and in adult zebrafish kidney cells (sorted by forward and side scatter charactersistics). Morpholinos (stable, synthesized antisense oligonucleotides that specifically block gene expression when injected into embryos at the one-cell stage) were designed to inhibit znpm1a or 1b and injected into zebrafish embryos at the 1–4 cells stage to assess the effect of knockdown of znpm1a and 1b alone and in combination on hematopoiesis. Whole-mount in situ hybridization of 48 hours post-fertilization(hpf) injected embryos demonstrated a 50% reduction in the expression of myeloperoxidase (mpo) and a similar reduction in alpha globin (α-globin) expression as markers of myelo- and erythropoiesis. To investigate the mechanism of the reduction in hematopoietic cells we injected the znpm1a and 1b morpholinos into zebrafish carrying mutated p53 and observed partial rescue of the hematopoietic phenotype suggesting that loss of npm1 in zebrafish activates p53 dependent cell cycle arrest, senescence or cell death. Thus zebrafish npm1 proteins are required for normal hematopoiesis consistent with the role for NPM1 as a tumor suppressor in AML/MDS with loss of all or part of chromosome 5. Future studies using this model will address which pathways are disrupted by the loss of npm1 and thus may contribute to the pathogenesis of human AML/MDS and facilitate identification of potential therapeutic targets. Figure 1. Zebrafish znpm1b exhibits synteny with human chromosome 5q35 and mouse chromosome 11. Figure 1. Zebrafish znpm1b exhibits synteny with human chromosome 5q35 and mouse chromosome 11.

Currently available combination chemotherapy for AML induces complete remission in about 75% of younger and 45% of older patients, but often fails to induce long-term disease-free survival and is toxic to normal hematopoietic cells. The frequent disease relapse observed in chemotherapy treated AML patients is thought to occur through the inability of the existing drugs to specifically target the self-renewing leukemia-initiating cells (LICs). These cells have the functional capacity to replenish AML blasts. An attractive target for AML therapy is the nuclear export protein exportin 1 (XPO1), which is functionally required for continuous nuclear export of a subset of proteins and mRNAs. Small molecule Selective Inhibitor of Nuclear Export (SINE) compounds covalently bind to Cysteine528 in the cargo-binding groove of XPO1 and inhibit nuclear export. The orally bioavailable clinical SINE compound, selinexor (KPT-330), is currently in Phase 1 and 2 clinical trials in adult patients with AML ([NCT01607892][1], [NCT02088541][2], [NCT02249091][3], [NCT02403310][4], [NCT02093403][5], [NCT02485535][6], [NCT02299518][7], [NCT02416908][8], [NCT02212561][9], and [NCT01607892][1]) and in a Phase 1 trial for relapsed childhood ALL and AML ([NCT02091245][10]). The results of these trials are encouraging, as they have demonstrated that selinexor alone or in combination is active in inducing remission in patients with relapsed or refractory AML. Here, we explore the anti-leukemia activity of a next generation SINE compound, KPT-8602. In preclinical toxicology studies as well as clinical trials, noted adverse events for selinexor are anorexia with weight loss, fatigue and thrombocytopenia, which limit the frequency of dosing to no more than every other day, three times a week. KPT-8602 is orally bioavailable and has similar pharmacokinetic properties to selinexor, with reduced brain penetration. Preliminary toxicology studies in rats and monkeys suggest that KPT-8602 has a substantially better tolerability profile, with reduced fatigue, thrombocytopenia, and anorexia compared to selinexor. Thus, KPT-8602 can be dosed orally on a daily basis for 5 days each week. The increased drug exposure with daily dosing and better tolerability of KPT-8602 leads directly to superior efficacy in pre-clinical models compared with selinexor. To define the anti-leukemia activity of KPT-8602 against primary AML blasts and LICs in a relevant pre-clinical setting, we established patient-derived xenograft (PDX) models, in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Mice engrafted with leukemic blasts were treated with vehicle, selinexor (20 mg/kg three times per week), or KPT-8602 (15 mg/kg daily for 4 weeks). KPT-8602 was highly active against blast cells from three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN), AML with complex karyotype (AML-CK), and MDS-derived AML (MDS/AML)), as evidenced by a reduction in leukemic engraftment in primary mice after treatment (Fig. 1A-C). At these doses, KPT-8602 demonstrated higher anti-leukemic activity than selinexor, with no leukemic cells detected in the bone marrow of two of the eight AML-CN PDX treated mice (Fig. 1A-C). In secondary transplantation assays, KPT-8602 greatly reduced the frequency of LICs in the PDX model derived from AML-CN cells, indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs (Fig. 1D). In mice engrafted with normal cord blood human CD34+ cells, KPT-8602 reduced bone marrow human CD45+ cell numbers (Fig. 1E), but the impact on normal hematopoietic stem and progenitor cells, as determined by secondary transplantation assays (Fig. 1E), was modest when compared to the LIC reduction after KPT-8602 treatment. These findings demonstrate that KPT-8602 has better tolerability, perhaps through decreased CNS penetration, and is better at eliminating both AML LIC and blast cells when compared to selinexor. Therefore, KPT-8602 should be tested for efficacy and tolerability in patients with relapsed and refractory AML, with the goal to overcome a difficult obstacle in curing AML; destroying the LIC compartment while sparing normal hematopoietic cells. ![Figure][11] Disclosures Etchin: Karyopharm: Research Funding. Baloglu: Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman: Karyopharm: Employment. Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Ellis: Karyopharm Therapeutics Inc: Employment. McCauley: Karyopharm Therapeutics, Inc: Employment. Stone: Agios: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Karyopharm: Consultancy; Celator: Consultancy; Novartis: Research Funding; Merck: Consultancy; Juno: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Roche/Genetech: Consultancy; Celgene: Consultancy. DeAngelo: Bristol Myers Squibb: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Ariad: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Kauffman: Karyopharm: Employment, Equity Ownership. Shacham: Karyopharm: Employment, Equity Ownership. Look: Karyopharm Therapeutics, Inc: Research Funding. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01607892&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02088541&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02249091&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [4]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02403310&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [5]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02093403&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [6]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02485535&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [7]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02299518&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [8]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02416908&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [9]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02212561&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [10]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02091245&atom=%2Fbloodjournal%2F126%2F23%2F326.atom [11]: pending:yes

Over the last decade, genetic characterization of pediatric T cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities, including translocations, deletions and amplifications. In this study, we used array-comparative genome hybridization (array-CGH) to identify a novel recurrent 9q34 amplification in 33 percent (12/36) of pediatric T-ALL samples, which is therefore one of the most frequent cytogenetic abnormalities observed in T-ALL thus far. The exact size of the amplified region differed slightly among patients, but the critical region involved VAV2, TRAF2 and NOTCH1. FISH analysis revealed that this 9q34 amplification was the result of a 9q34 duplication on one chromosome and could be identified in 17 to 39 percent of the leukemic cells at diagnosis. Although the presence of this leukemic subclone did not predict for poor clinical outcome in our small patient cohort, leukemic cells carrying this duplication were still present at times of relapse, indicating that these cells effectively survived intensive chemotherapeutic treatment. Episomal NUP214-ABL1 amplification and activating mutations in NOTCH1, two other recently identified 9q34 abnormalities in T-ALL, were also detected in our pediatric patient cohort. We showed that both genetic abnormalities are independent from this newly identified 9q34 duplication.

Recent studies have demonstrated that most patients with T-cell acute lymphocytic leukemia (T-ALL) have activating mutations in NOTCH1. We sought to determine whether these mutations are also acquired in mouse models of T-ALL. We sequenced the heterodimerization domain and the PEST domain of Notch1 in our mouse model of TAL1-induced leukemia and found that 74% of the tumors harbor activating mutations in Notch1. Cell lines derived from these tumors undergo G0/G1 arrest and apoptosis when treated with a γ-secretase inhibitor. In addition, we found activating Notch1 mutations in 31% of thymic lymphomas that occur in mice deficient for various combinations of the H2AX, Tp53, and Rag2 genes. Thus, Notch1 mutations are often acquired as a part of the molecular pathogenesis of T-ALLs that develop in mice with known predisposing genetic alterations. (Blood. 2006;107:781-785)

Activating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML)/atypical chronic myelogenous leukemia (aCML), myelodysplastic syndrome (MDS), B-lineage acute lymphoblastic leukemia (ALL), T-cell ALL, and chronic lymphocytic leukemia (CLL). Analysis of 222 patients with AML identified JAK2V617F mutations in 4 patients with AML, 3 of whom had a preceding MPD. JAK2V617F mutations were identified in 9 (7.8%) of 116 CMML/a CML samples, and in 2 (4.2%) of 48 MDS samples. We did not identify the JAK2V617F disease allele in B-lineage ALL (n = 83), T-cell ALL (n = 93), or CLL (n = 45). These data indicate that the JAK2V617F allele is present in acute and chronic myeloid malignancies but not in lymphoid malignancies.

The zebrafish is an attractive vertebrate model for genetic studies of development, apoptosis, and cancer. Here we describe a transgenic zebrafish line in which T- and B-lymphoid cells express a fusion transgene that encodes the zebrafish bcl-2 protein fused to the enhanced green fluorescence protein (EGFP). Targeting EGFP-bcl-2 to the developing thymocytes of transgenic fish resulted in a 2.5-fold increase in thymocyte numbers and a 1.8-fold increase in GFP-labeled B cells in the kidney marrow. Fluorescent microscopic analysis of living rag2-EGFP-bcl-2 transgenic fish showed that their thymocytes were resistant to irradiation- and dexamethasone-induced apoptosis, when compared with control rag2-GFP transgenic zebrafish. To test the ability of bcl-2 to block irradiation-induced apoptosis in malignant cells, we compared the responsiveness of Myc-induced leukemias with and without EGFP-bcl-2 expression in living transgenic zebrafish. T-cell leukemias induced by the rag2-EGFP-Myc transgene were ablated by irradiation, whereas leukemias in double transgenic fish expressing both Myc and EGFP-bcl-2 were resistant to irradiation-induced apoptotic cell death. The forward genetic capacity of the zebrafish model system and the ability to monitor GFP-positive thymocytes in vivo make this an ideal transgenic line for modifier screens designed to identify genetic mutations or small molecules that modify bcl-2-mediated antiapoptotic pathways. (Blood. 2005;105:3278-3285)

The study of hematopoiesis has been greatly facilitated by transplantation of blood cell populations into recipient animals. Efficient engraftment of donor cells generally requires ablation of the host hematopoietic system. The zebrafish has recently emerged as a developmental and genetic system to study hematopoiesis. To enable the study of hematopoietic stem cell (HSC) biology, immune cell function, and leukemogenesis in zebrafish, we have developed hematopoietic cell transplantation (HCT) into adult recipient animals conditioned by γ irradiation. Dose-response experiments showed that the minimum lethal dose (MLD) of 40 Gy led to the specific ablation of hematolymphoid cells and death by 14 days after irradiation. Sublethal irradiation doses of 20 Gy predominantly ablated lymphocytes and permitted transplantation of a lethal T-cell leukemia. Finally, transplantation of hematopoietic cells carrying transgenes yielding red fluorescent erythrocytes and green fluorescent leukocytes showed that HCT is sufficient to rescue the MLD, that recipient hematolymphoid tissues were repopulated by donor-derived cells, and that donor blood cell lineages can be independently visualized in living recipients. Together, these results establish transplantation assays to test for HSC function and oncogenic transformation in zebrafish.

In pro-B cell acute lymphoblastic leukemia (ALL), expression of the E2A-HLF fusion gene as a result of t(17;19)(q22;p13) is associated with poor prognosis, hypercalcemia, and hemorrhagic complications. We previously reported that the E2A-HLF fusion protein protects interleukin-3 (IL-3)–dependent lymphoid cells from apoptosis caused by cytokine starvation. Here, we report that annexin II, a surface phospholipid-binding protein and one of the proposed causes of the hemorrhagic complications of acute promyelocytic leukemia (APL), is also implicated in t(17;19)+ ALL. Annexin II was expressed at high levels in APL cells and in each of 4 t(17;19)+ leukemia cell lines, and annexin II expression was induced by enforced expression of E2A-HLF in leukemia cells. In IL-3–dependent cells, we found that annexin II expression was regulated by IL-3 mainly by Ras pathways, including Ras/phosphatidylinositol 3-kinase pathways. Moreover, E2A-HLF increased annexin II expression in IL-3–dependent cells in the absence of the cytokine. These findings indicate that E2A-HLF induces annexin II by substituting for cytokines that activate downstream pathways of Ras. (Blood. 2004;103:3185-3191)

Activating FLT3 mutations are the most common genetic aberrations in acute myeloid leukemia (AML), resulting in the constitutive activation of this receptor tyrosine kinase (RTK), but such mutations are rarely found in acute lymphoblastic leukemia (ALL). Here we describe a unique subset of de novo adult T-cell ALL (T-ALL) cases that coexpress CD117/KIT and cytoplasmic CD3 (CD117/KIT+ ALL). Activating mutations in the FLT3 RTK gene were found in each of 3 CD117/KIT+ cases that were analyzed, but not in 52 other adult T-ALL samples from the same series that lacked CD117/KIT expression. Our results indicate the need for clinical trials to test the efficacy of drugs that inhibit the FLT3 RTK in this subset of patients with T-ALL. (Blood. 2004;104:558-560)

The specific targeting of critical signaling molecules may provide efficient therapies for T-cell acute lymphoblastic leukemia (T-ALL). However, target identification and drug development are limited by insufficient numbers of primary T-ALL cells and by their high rate of spontaneous apoptosis. We established a human interleukin-7 (IL-7)–dependent T-ALL cell line, TAIL7, that maintains several biologic and signaling properties of its parental leukemia cells. TAIL7 cells are pre–T-ALL cells that proliferate in response to IL-7 and IL-4. IL-7 stimulation of TAIL7 cells prevents spontaneous in vitro apoptosis and induces cell activation and cell cycle progression. The signaling events triggered by IL-7 include down-regulation of p27kip1 and hyperphosphorylation of retinoblastoma protein (Rb). Stimulation of TAIL7 cells by IL-7 leads to phosphorylation of Janus kinase 3 (JAK3), signal transducer and activator of transcription 5 (STAT5), Akt/PKB (protein kinase B), and extracellular-regulated kinase 1 and 2 (Erk1/2). Importantly, specific blockade of JAK3 by its inhibitor WHI-P131 abrogates the IL-7–mediated proliferation and survival of TAIL7 cells, suggesting that activation of JAK3 is critical for IL-7 responsiveness by these cells. Because TAIL7 cells seem to be a biologic surrogate for primary leukemia T cells, this cell line constitutes a valuable tool for the study of the signaling pathways implicated in T-ALL. Exploitation of this cell line should allow the identification of molecular targets and promote the rational design and validation of antileukemia signaling inhibitors.

Aberrant expression of transcription factor oncogenes such as HOX11, HOX11L2, TAL1/SCL, LYL1, LMO1, and LMO2 can be detected in lymphoblasts from up to 80% of patients with acute T-cell lymphoblastic leukemia (T-ALL). Transcriptional activation of these oncogenes in leukemic cells typically results from chromosomal rearrangements that place them next to highly active cis-acting transcriptional regulatory elements. However, biallelic activation of TAL1 in some T-ALL cases has been previously proposed. We have used allele-specific mRNA analysis to show that trans-acting mechanisms leading to biallelic overexpression of TAL1 are involved in 10 (42%) of 24 TAL1+ informative T-ALL cases, 2 (17%) of 12 HOX11+ informative cases, and 7 (64%) of 11 LMO2+ informative cases. We propose that aberrant expression of oncogenic transcription factors in a significant fraction of T-ALLs may result from loss of the upstream transcriptional mechanisms that normally down-regulate the expression of these oncogenes during T-cell development.

Currently available combination chemotherapy for acute myeloid leukemia (AML) often fails to induce long-term remissions, prompting the need to develop novel therapies. The frequent disease relapse that is observed in patients with AML is thought to occur because of the inability of the existing drugs to target the self-renewing leukemia-initiating (LICs) in AML. An attractive new strategy for AML therapy is inhibition of the nuclear export protein exportin 1 (XPO1), also called CRM1. A member of the karyopherin b family, XPO1 mediates nuclear export of proteins that contain leucine-rich nuclear export signals (NES), including protein adaptors that transport RNA molecules. XPO1 regulates nuclear export of ~220 eukaryotic proteins, including the tumor suppressor proteins p53, p73, p21, Rb, FOXO3A, BRCA1, and PP2A, cell cycle regulators, and apoptotic proteins. Indeed, nuclear-cytoplasmic transport by XPO1 is required for the survival of several types of solid tumors and hematologic malignancies. Recently, novel selective small molecule inhibitors of nuclear export (SINE) that inhibit the export function of XPO1 by targeting Cys528 in its NES-binding groove, were developed using an in silico molecular modeling strategy. The orally bioavailable SINE compound, selinexor (KPT-330), is in Phase 1 and 2 studies in patients with adult AML ([NCT01607892][1] and [NCT02088541][2]) and in a Phase 1 study for relapsed childhood ALL and AML that was initiated in March, 2014 ([NCT02091245][3]). To determine the anti-leukemic activity of selinexor against primary AML blasts and LICs in a clinically relevant setting, we established mouse xenograft models of human leukemia, in which leukemic blast cells from patients with different subtypes of AML were transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Engrafted mice were treated with selinexor or a vehicle control. Selinexor was highly active against blast cells from two of the three patients with poor-prognosis disease (cytogenetically normal AML with an internal tandem duplication of FLT3 (AML-CN) and complex karyotype AML (AML-CK1 and AML-CK2)) as evidenced by a reduction in leukemic engraftment in primary mice. Importantly, serial dilution transplantation assays indicate that selinexor therapy greatly reduced the frequency of LICs in xenografts derived from all three patients (6 - to 434- fold reduction compared to controls), indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs. Interestingly, in mice bearing AML grafts derived from one of the patients with complex karyotype, selinexor dramatically decreased LIC frequency despite exhibiting only modest anti-leukemic activity against bulk disease in primary mice. These findings show that selinexor has potent activity against self-renewing LIC in poor-prognosis AML, even when it has only moderate activity against the bulk AML cell population. Moreover, bone marrow biopsies of selinexor-treated mice showed normal hematopoietic cell morphology and cellularity following four weeks of treatment. These findings demonstrate that inhibition of nuclear export with selinexor addresses the primary remaining problem in the therapy of AML, which is to destroy the very critical self-renewing LIC compartment while sparing normal hematopoietic cells. Disclosures Etchin: Leukemia and Lymphoma Society, Alex's Lemonade Stand, Luck2Tuck Foundation, Karyopharm Therapeutics, Inc: Research Funding. McCauley: Karyopharm Therapeutics, Inc: Employment, Equity Ownership. Kauffman: Karyopharm Therapeutics, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Shacham: Karyopharm Therapeutics, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01607892&atom=%2Fbloodjournal%2F124%2F21%2F995.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02088541&atom=%2Fbloodjournal%2F124%2F21%2F995.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02091245&atom=%2Fbloodjournal%2F124%2F21%2F995.atom

Epigenetic therapy implies the use of drugs that target regulators of gene expression, such as DNA and histone methylation or histone acetylation, without altering the DNA coding sequence. Epigenetic mechanisms have been found to be perturbed in myeloid diseases, and in fact, the DNA demethylating agents, 5-azacytadine and decitabine are FDA approved drugs for myelodysplastic syndrome. However, use of epigenetic therapies in other types of myeloid disease including acute myeloid leukemia (AML) has been met with variable success, suggesting efficacy might be improved by preselecting subtypes of disease in which there is a high degree of epigenetic dysregulation. AML has recently been found to be associated with a host of epigenetic abnormalities including mutations in DNA methyltransferase 3A (DNMT3A), ten-eleven translocation-2 (TET2) and enhancer of zeste homolog 2 (EZH2). We generated a transgenic zebrafish model of high risk myeloid disease expressing the human NUP98-HOXA9 (NHA9) fusion oncogene, a genetic lesion for which epigenetic dysregulation has not previously been identified. Transgenic embryos exhibit an increase in immature myeloid cells at the expense of erythroid cells and adult fish develop a myeloproliferative neoplasm (MPN). Leveraging this model in a microarray screen, we identified 3-fold elevated levels of the epigenetic regulator, dnmt1, the major maintenance methyltransferase, for the first time in high risk AML. Decitabine specifically inhibits DNMT1 and treatment of NHA9 transgenic embryos with 75µM decitabine restored normal hematopoiesis, as evidenced by normal numbers of leukocytes and red cells. Moreover, using hematopoietic stem cell (HSC) reporter lines and whole mount in situ hybridization, we identified a 2-3 fold increase in this population in NHA9 embryos, suggesting the HSC as the cell of origin in this disease. Interestingly, 75µM decitabine therapy also restored normal HSC numbers. Strikingly, we discovered synergy when we combined sub-monotherapeutic doses of DNMT1 inhibitors, decitabine (10-25µM) or zebularine (100µM), plus histone deacetylase inhibitors, valproic acid (25-100µM) or trichostatin A (250nM), to inhibit the effects of NHA9 on hematopoiesis. To determine if NHA9 expression directly results in changes to DNA methylation, we performed MeDIP-Seq on genomic DNA from pools of untreated and decitabine-treated NHA9 or control embryos. Untreated NHA9 embryos displayed significantly higher methylation levels at the regions of gene promoters compared to control embryos, which were restored to control levels following treatment with 75µM decitabine. Similarly, combination therapy with 10µM decitabine and 25µM valproic acid significantly reduced methylation to near control levels following a 5 hour treatment, while prolonged exposures to these same doses resulted in profound global hypomethylation. These findings elucidate underlying mechanisms in the pathogenesis of NHA9-induced myeloid disease and propose novel actionable epigenetic drug targets. Furthermore, we highlight the opportunities inherent in the zebrafish model as a unique in vivo platform for the preclinical screening of epigenetic based combination therapy. Disclosures No relevant conflicts of interest to declare.