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The tyrosine kinase JAK3 plays a well-established role during normal lymphocyte development and is constitutively phosphorylated in several lymphoid malignancies. However, its contribution to lymphomagenesis remains elusive. In this study, we used the newly identified activating JAK3A572V mutation to elucidate the effect of constitutive JAK3 signaling on murine lymphopoiesis. In a bone marrow transplantation model, JAK3A572V induces an aggressive, fatal, and transplantable lymphoproliferative disorder characterized by the expansion of CD8+TCRαβ+CD44+CD122+Ly-6C+ T cellsthat closely resemble an effector/memory T-cell subtype. Compared with wild-type counterparts, these cells show increased proliferative capacities in response to polyclonal stimulation, enhanced survival rates with elevated expression of Bcl-2, and increased production of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα), correlating with enhanced cytotoxic abilities against allogeneic target cells. Of interest, the JAK3A572V disease is epidermotropic and produces intraepidermal microabscesses. Taken together, these clinical features are reminiscent of those observed in an uncommon but aggressive subset of CD8+ human cutaneous T-cell lymphomas (CTCLs). However, we also observed a CD4+ CTCL-like phenotype when cells are transplanted in an MHC-I–deficient background. These data demonstrate that constitutive JAK3 activation disrupts T-cell homeostasis and induces lymphoproliferative diseases in mice.

To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways.

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5′RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)–independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.

Abstract 384 The Notch signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment to a specific lineage is unclear. During hematopoiesis, the Notch signaling pathway is known to play an important role in T cell lineage development. Recently, we demonstrated that the Notch signaling pathway is also a positive regulator of megakaryocyte lineage specification from hematopoietic stem cells (HSC). The importance of a tight regulation of this latter role is highlighted by the aberrant activation of the canonical Notch pathway transcription factor RBPJ by OTT-MAL, a fusion oncogene specifically associated with infant acute megakaryoblastic leukemia (AMKL). Here, we report a crosstalk between the Notch and PI3K/AKT pathways that provides new insights into the mechanism through which Notch signaling pathway regulates HSC differentiation into the erythro-megakaryocytic lineages. First, we observed that cells expressing a constitutively active Notch mutant had an increased level of phosphorylation of AKT compared to controls, indicating an association between Notch and AKT pathway activation. Using a Notch-GFP reporter mouse line, we confirmed that phosphorylation of AKT was increased in wild-type bone marrow cells upon physiological Notch stimulation (i.e. GFP+ cells) compared to control cells (i.e. GFP- cells) in vivo. Next, we assessed whether PI3K/AKT activation could replace or mimic Notch signaling during megakaryocyte development by transducing Lineage-Sca-1+cKit+ (LSK) cells or committed common myeloid progenitors (CMP) with a constitutively activated myristoylated AKT (myrAKT) mutant, followed by plating with or without Notch pathway stimulation on OP9-DL1 stroma or OP9 control stroma, respectively. MyrAKT-expressing LSK cells did not efficiently give rise to CD41+ megakaryocytic cells in the absence of Notch pathway stimulation, whereas myrAKT-expressing CMP showed partial rescue of development of megakaryocytes. Conversely, expression of a kinase-dead AKT mutant resulted in a pronounced reduction in megakaryocyte development from CMP, but had only a modest effect on LSK differentiation. Similar results were obtained with a chemical inhibitor of the AKT pathway. These results indicate that PI3K/AKT activation acts as an essential effector of the Notch pathway and can mimic Notch stimulation in CMP, whereas Notch-induced megakaryopoiesis from LSK cells is largely independent of the status of the PI3K/AKT pathway. To investigate the role of PI3K-AKT pathway on megakaryocyte development in vivo, we used FoxO1/3/4-deficient and PTEN-deficient mice, and observed that both mouse lines had significantly increased megakaryopoiesis compared to control animals both in vivo and ex vivo after culture on OP9-DL1 stroma. Importantly, FoxO1/3/4-deficient progenitors had upregulation of Nrarp and Hes1, two Notch pathway targets, and chromatin immunoprecipitation assays revealed the presence of FoxO factors at the Hes1 promoter, indicating a feedback control of the PI3K/AKT pathway on Notch pathway activation. Taken together, these data demonstrate a complex regulatory network between the Notch and PI3K/AKT pathways during megakaryopoiesis. In addition, our results annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of two distinct developmental pathways. Disclosures: Gilliland: Merck: Employment.

The Notch signaling pathway is involved in a broad spectrum of cell fate decisions during development, and in the hematopoietic system, it is known to favor T cell- vs B cell lineage commitment. However, its role in myeloid lineage development is less well understood. We have shown, using heterotypic co-cultures of murine primary hematopoietic stem cells (Lin-Sca-1+ckit+ HSCs) and OP9 stromal cells expressing the Notch ligand Delta1 (OP9-DL1), that Notch signaling derived from cell non-autonomous cues acts as a positive regulator of megakaryocyte fate from LSK cells. Bone marrow transplantation experiments with a constitutively active Notch mutant resulted in enhanced megakaryopoiesis in vivo, with increased MEP numbers and megakaryocyte colony formation. In contrast, expression of dnMAML using a conditional ROSA26 knock-in mouse model significantly impaired megakaryopoiesis in vivo, with a marked decrease in megakaryocyte progenitors. In order to understand the cellular differentiation pathways controlled by Notch, we first examined the ability of various purified progenitor populations to differentiate toward megakaryocytes upon Notch stimulation in vitro. We observed that CMP and MEP, but not GMP, can engage megakaryopoiesis upon Notch stimulation. Our results were consistent with expression analysis of Notch signaling genes in these purified progenitors and were supported by the observation that transgenic Notch reporter mice display higher levels of reporter (i.e. GFP) expression in HSC and MEP, vs. CMP and GMP in vivo. Furthermore, purified progenitors with high GFP expression gave rise to increased numbers of megakarocyte-containing colonies when plated in vitro compared to GFP-negative progenitors. In addition, further purification of the HSC population into long-term (LT), short-term (ST), and lymphoid-primed myeloid progenitors (LMPP) before plating on OP9-DL1 stroma showed that LMPP have a reduced ability to give rise to megakaryocytes compared to the other two populations. These data support the hypothesis that there is an early commitment to erythro/megakaryocytic fate from HSC prior to lymphoid commitment. To gain insight into the molecular mechanism underlying Notch-induced megakaryopoiesis, we performed global gene expression analysis that demonstrated the engagement of a megakaryopoietic transcriptional program when HSC were co-cultured with OP9-DL1 vs. OP9 stroma or OP9-DL1 treated with gamma-secretase inhibitor. Of interest, Runx1 was among the most upregulated genes in HSC co-cultured on OP9-DL1 stroma. To assess whether Notch signaling engages megakaryocytic fate through induction of Runx1, we plated HSC from Runx1 −/− mice on OP9-DL1 stroma. Compared to WT cells, Runx1 −/− HSC had a severely reduced ability to develop into CD41+ cells. In contrast, overexpression of Runx1 in WT HSC was sufficient to induce megakaryocyte fate on OP9 stroma without Notch stimulation. Together, our results indicate that Notch pathway activation induced by stromal cells is an important regulator of cell fate decisions in early progenitors. We show that Notch signaling is upstream of Runx1 during Notch-induced megakaryocyte differentiation and that Runx1 is an essential target of Notch signaling. We believe that these results provide important insight into the pathways controlling megakaryocyte differentiation, and may have important therapeutic potential for megakaryocyte lineage-related disorders.

The Notch pathway regulates a broad range of biological mechanisms including proliferation, border formation and cell fate decisions. In the hematopoietic system, Notch signaling is generally thought to specify T cell lineage fate at the expense of the B cell whereas its role in the myeloid lineage development is unclear. When using heterotypic co-cultures of murine primary hematopoietic stem cells (HSC: Lin-Sca1+Kit+) with OP9 stromal cells, or OP9 cells expressing the Notch ligand Delta1 (OP9-DL1), we unexpectedly observed the development of large cells with cytoplasmic protrusions reminiscent of proplatelet production by megakaryocytes on OP9-DL1 stroma. These cells stained positive for acetylcholinesterase, specific for megakaryocyte, and displayed large polylobated nuclei. Flow cytometric analysis indicated a 10-fold increase in the number of CD41+ cells in OP9-DL1 co-cultures compared to parental OP9 co-cultures. Expression of a constitutively active intra-cellular Notch (ICN) mutant allowed differentiation of HSC into CD41+ cells in parental OP9 co-cultures without DL1 stimulation, whereas expression of a dominant-negative MAML1 (dnMAML1) mutant abrogated this effect in OP9-DL1 co-cultures. In addition, megakaryocyte differentiation in OP9-DL1 co-cultures was blocked by γ-secretase inhibitors treatment and rescued by ectopic expression of ICN. Global gene expression analysis demonstrated engagement of a megakaryopoietic transcriptional program when HSC were co-cultured with OP9-DL1 vs. OP9 stroma or OP9-DL1 stroma treated with γ-secretase inhibitor. Bone marrow transplantation experiments with ICN, resulted in enhanced megakaryopoiesis in vivo with increased MEP numbers and megakaryocyte colony formation. Furthermore, transplantation of bone marrow cells transduced with dnMAML1 significantly impaired megakaryopoiesis in vivo with a 4- to 7-fold decrease in maturing megakaryocytes. These findings demonstrate a positive regulatory role for Notch signaling in specification of megakaryocyte development, and indicate that Notch plays a complex role in cell fate decisions among myeloid progenitors. They suggest the possibility that inhibition of Notch signaling may have therapeutic potential in malignancies of the megakaryocytic lineage. Furthermore, Notch pathway stimulation could be of value in enhancing megakaryocyte growth in clinical contexts associated with severe thrombocytopenia, such as hematopoietic reconstitution following bone marrow transplantation or chemotherapy.

Molecular pathogenesis of human peripheral T-cell malignancies remains poorly understood. We aimed to establish a model for these disorders by using the recently identified JAK3A572V allele. This mutation, which lies in the pseudokinase (JH2) domain of JAK3, transforms Ba/F3 cells to factor-independent growth and causes a striking lymphoproliferative phenotype in a murine bone marrow transplantation assay. Further characterization of JAK3A572V animals revealed that the aberrantly expanded cell population consisted of a mature effector/memory subtype of CD8+T-cells that infiltrated all major lymphoid and several non-lympoid organs and could be transplanted into secondary and tertiary recipients. These JAK3A572V T-cells had increased proliferative capacities and displayed enhanced phosphorylation of common JAK3 targets, such as STAT5 and S6-kinase. Proliferation of primary T-cells transformed by JAK3A572V was effectively inhibited with a small molecule JAK inhibitor that had no effect on the proliferative potential of control cells transduced with a wildtype JAK3 allele. Furthermore, the mutant cells showed increased production of cytotoxic cytokines, such as IFN-γ and TNF-α, compared to wildtype counterparts, which correlated with an increased cellular cytotoxicity towards allogeneic target cells. Of particular interest, JAK3A572V animals presented with skin lesions and histopathologic analysis showed aberrant skin-homing T-cells tagging along the epidermal/dermal junctions. Mice receiving Rag1-deficient donor cells transduced with the JAK3A572V allele also developed a lethal lymphoproliferative disease characterized by the expansion of immature CD3−TCRβ−CD4+/−CD8+ cells, suggesting that the JAK3A572V-dependent lymphoproliferation does not require proper TCR rearrangement. Altogether these results indicate that in this murine model, constitutive activation of JAK3 results in peripheral/cutaneous T-cell lymphoma (PTCL) that closely resembles the human disease. These findings suggest the possibility that the molecular basis of human PTCL could include aberrant JAK3 signaling and might provide a useful platform for deciphering the molecular and cellular mechanisms and requirements for peripheral lymphoid disease development and progression. Furthermore, it provides an opportunity to investigate the therapeutic potential of selective JAK3 inhibitors for this subset of lymphoid disorders, whose treatment remains a challenge.

Notch signaling represents a central pathway regulating hematopoiesis, stem cell differentiation, and malignant transformation in human cancer. Activation of highly conserved Notch1 receptors results in cleavage and release of an intracellular domain (ICN1). Following translocation to the nucleus, ICN1 forms a ternary complex with the transcriptional repressor CSL (CBF-1, Suppressor of Hairless and Lag-1) bound to cognate DNA. This event triggers a repressor-to-activator switch, as an interfacial groove is formed which recruits the Mastermind-Like (MAML1) co-activator protein. Activating mutations in NOTCH1 are found in more than 50% of patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL), promoting protein stability and establishing a direct link to disease pathogenesis. Pharmacologic efforts to target the Notch pathway in T-ALL have been directed at gamma secretase, a regulatory enzyme in Notch activation. Recently, the observed clinical resistance to gamma secretase inhibitors has been explained, in part, by additional mutations in the Notch-targeting ubiquitin ligase, Fbxw7, which further increases oncoprotein stability. Therefore, direct inhibitors of ICN1 function are highly desirable. Drawing upon insights afforded by the resolved crystal structure of the DNA-bound ICN1:MAML1:CSL complex, we synthesized a series of hydrocarbon stapled alpha-helical peptides targeting Notch (SAHNs) based on minimal motifs of the MAML protein predicted to engage the composite ICN1:CSL interface. Direct, high-affinity binding to purified components of the Notch complex was confirmed using surface plasmon resonance (SPR). Nuclear access of SAHN1 was confirmed using quantitative epifluorescent and confocal microscopy. Intracellular association with ICN1 and CSL was established using bidirectional affinity chromatography. Using a novel CSL-responsive reporter construct, we observed inhibition of endogenous Notch transactivation by SAHN1 in T-ALL cell lines. Furthermore, SAHN1 induces a dose-dependent knockdown of endogenous Notch1 target genes including HES1, HEY1 and cMYC in T-ALL cell lines. Remarkably, inhibition of Notch signaling by SAHN1 confers selective cytotoxicity at 48 hours in a panel of T-ALL cell lines with known mutations in NOTCH, including those resistant to gamma secretase inhibitors. Supporting an on-target mechanism of action, we have prepared a damaged analogue of SAHN1 containing a two-residue rearrangement (SAHN1D). SAHN1D possesses reduced binding affinity for the Notch complex and despite comparable intracellular access, SAHN1D lacks both transcriptional and cytotoxic effects on cultured T-ALL cell lines in vitro. Efficacy studies have also been performed in vivo using a novel murine model of T-ALL. In summary, we report here the design, biochemical characterization and translational rationale supporting the first direct inhibitor of the Notch transactivation complex in T-ALL.

Leukemias are often associated with aberrant tyrosine kinase activity that occurs as a result of chromosomal translocations. These mutations are able to confer a proliferative and survival advantage to leukemic cells, as well as cooperate with other mutations that impair cell differentiation, thus leading to the development of leukemia. NUP214-ABL1 is one such recently identified fusion gene that is generated by episomal amplification. The presence of the fusion was recently identified in approximately 6% of patients with T-cell acute lymphoblastic leukemia (T-ALL). By the use of a murine retroviral bone marrow transplantation model we have demonstrated that mice transplanted with NUP214-ABL1 transduced bone marrow cells developed either a myeloproliferative disorder (MPD) with a disease latency of 70 to 118 days or a T cell lymphoblastic leukemia with a disease latency of 115 to 124 days. The myeloproliferative phenotype was characterized by splenomagaly and leukocytosis, and analysis of the histopathology revealed extramedullary hematopoiesis in the liver, lung, kidney and Peyer’s patches, and an increase of peripheral blood neutrophils. Flow cytometry of single cell suspensions from spleen and bone marrow samples of mice with a myeloproliferative phenotype demonstrated an increase of Gr-1+/Mac-1+ cells (approximately 70%). Two of the mice that were transplanted with NUP214-ABL1 transduced bone marrow cells developed T cell lymphomas that were characterized by large thymomas, a phenotype that is consistent with other models of activated tyrosine kinases over long disease latencies. Histopathological analysis of the thymi revealed effacement of normal thymic architecture as well as T cell infiltrate into the surrounding skeletal muscle. In addition, flow cytometric analysis revealed a significant increase in the CD4+/CD8+ T cell population in the thymi of these animals. No disease was observed in secondary transplant recipients following 60 days of observation. In conclusion, these results indicate that NUP214-ABL1 is able to cause either a myeloproliferative disease or a T cell lymphoma over longer latencies in mice, the latter being similar to the phenotype observed in humans with expression of the NUP214-ABL1 fusion. These findings provide a useful model for future experiments to determine if there is a contribution of other mutations together with the NUP214-ABL1 fusion towards the development of a T-ALL phenotype in mice.