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Dysregulation of the BCL-2 family is implicated in protecting chronic myeloid leukemia (CML) cells from intracellular damage and BCR::ABL1-inhibition with tyrosine kinase inhibitors (TKIs) and may be a viable therapeutic target in blast phase (BP-)CML, for which treatment options are limited. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic and acute myeloid leukemia as single agents and in combination with standard-of-care therapies. Here we present the first comparison of inhibition of BCL-2 prosurvival proteins BCL-2, BCL-xL and MCL-1 in combination with a second or third generation TKI, crucially with comparisons drawn between myeloid and lymphoid BP-CML samples. Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Nilotinib with BH3 mimetic combinations in myeloid BP-CML patient samples triggered increased induction of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and CD34+ fraction, while this was not the case for lymphoid BP-CML samples tested. While some heterogeneity in apoptotic response was observed between cell lines and BP-CML patient samples, the combination of BCL-xL and BCR::ABL1 inhibition was consistently effective in inducing substantial apoptosis. Further, while BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically induced apoptosis in all cell lines tested and in myeloid BP-CML patient samples compared to healthy donor samples. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought.

Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6-8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML.

Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6–8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML.

Acute Myeloid Leukaemia (AML) is a commonly occurring severe haematological malignancy, with most patients exhibiting sub-optimal clinical outcomes. Therapy resistance significantly contributes towards failure of traditional and targeted treatments, disease relapse and mortality in AML patients. The mechanisms driving therapy resistance in AML are not fully understood, and approaches to overcome therapy resistance are important for curative therapies. To date, most studies have focused on therapy resistant mechanisms inherent to leukaemic cells (e.g., TP53 mutations), overlooking to some extent, acquired mechanisms of resistance through extrinsic processes. In the bone marrow microenvironment (BMME), leukaemic cells interact with the surrounding bone resident cells, driving acquired therapy resistance in AML. Growing evidence suggests that macrophages, highly plastic immune cells present in the BMME, play a role in the pathophysiology of AML. Leukaemia-supporting macrophage subsets (CD163+CD206+) are elevated in preclinical in vivo models of AML and AML patients. However, the relationship between macrophages and therapy resistance in AML warrants further investigation. In this review, we correlate the potential links between macrophages, the development of therapy resistance, and patient outcomes in AML. We specifically focus on macrophage reprogramming by AML cells, macrophage-driven activation of anti-cell death pathways in AML cells, and the association between macrophage phenotypes and clinical outcomes in AML, including their potential prognostic value. Lastly, we discuss therapeutic targeting of macrophages, as a strategy to circumvent therapy resistance in AML, and discuss how emerging genomic and proteomic-based approaches can be utilised to address existing challenges in this research field.

Execution of the intrinsic apoptotic pathway is controlled by the BCL-2 proteins at the level of the mitochondrial outer membrane (MOM). This family of proteins consists of prosurvival (e.g., BCL-2, MCL-1) and proapoptotic (e.g., BIM, BAD, HRK) members, the functional balance of which dictates the activation of BAX and BAK. Once activated, BAX/BAK form pores in the MOM, resulting in cytochrome c release from the mitochondrial intermembrane space, leading to apoptosome formation, caspase activation, and cleavage of intracellular targets. This pathway is induced by cellular stress including DNA damage, cytokine and growth factor withdrawal, and chemotherapy/drug treatment. A well-documented defense of leukemia cells is to shift the balance of the BCL-2 family in favor of the prosurvival proteins to protect against such intra- and extracellular stimuli. Small molecule inhibitors targeting the prosurvival proteins, named ‘BH3 mimetics’, have come to the fore in recent years to treat hematological malignancies, both as single agents and in combination with standard-of-care therapies. The most significant example of these is the BCL-2-specific inhibitor venetoclax, given in combination with standard-of-care therapies with great success in AML in clinical trials. As the number and variety of available BH3 mimetics increases, and investigations into applying these novel inhibitors to treat myeloid leukemias continue apace the need to evaluate where we currently stand in this rapidly expanding field is clear.

Background: Acute myeloid leukemia (AML) arises due to an accumulation of genetic lesions within myeloid progenitors and oncogenic transformation is often characterised by disordered transcription. Recently the histone lysine demethylase KDM4A was shown to be essential for AML blast survival and self-renewal. shRNA knockdown (KD) of KDM4A led to downregulated expression of the transcription factor NFATC2 an MLL-AF9 AML model, suggesting that it is a key target of KDM4A oncogenic function. The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors control cell cycle genes and self-renewal pathways in hematopoietic tissues and are well-defined as oncogenic regulators in various malignancies. NFATs have recently been attributed roles in the development of FLT3 ITD AML and resistance to tyrosine kinase inhibitors (TKIs) in myeloid leukemias but there is little evidence detailing the role(s) of NFATC2 specifically in AML. We hypothesized that NFATc2 activity is essential for the survival of AML cells and the oncogenic transcriptional networks within these. Aims: To determine if AML cells are dependent on NFATC2 for survival and to elucidate the transcriptional and binding targets of NFATc2 in AML. Methods: NFATC2 was depleted using shRNA KD in numerous cell line models of AML and putative transcriptional targets were elucidated using RNA-seq following KD. Binding targets of NFATc2 were determined using ChIP-seq. Transcriptomic targets of NFATc2 were validated using the Fluidigm Biomark multiplex PCR system and real time quantitative PCR. Results: KD of NFATC2 significantly impaired the colony forming capacity and expansion in liquid cultures of AML cell lines from diverse (cyto)genetic backgrounds. MLL-AF9/TP53 mut THP-1 cells showed reduced entry to the S-phase of the cell cycle and downregulation of cyclin D1 following NFATC2 depletion, suggesting that NFATC2 is critical for cell cycle progression in these cells. Overexpression of human NFATC2 in THP-1 led to an increased rate of cell growth. RNA-seq analysis of THP-1 cells with NFATC2 KD revealed >20 genes with deregulated expression (FDR ChIP-seq analysis of NFATc2 binding in THP-1 cells showed that >30% of NFATc2 targets were at promoter regions within 5kb of the transcription start site. Motif analysis of precipitated DNA fragments discovered two novel motifs which were enriched at NFATc2 binding sites (p Discussion: NFATC2 was found to be essential for expansion of AML cells in various cell line models. In the MLL-AF9 driven THP-1 model a number of putative transcriptional and genomic targets were defined, which include novel targets not previously described in AML pathogenesis and targets of MYC, an established oncogenic protein in AML. The differing expression profiles observed across AML cell lines of diverse (cyto)genetic backgrounds with NFATC2 KD suggest that the regulatory targets of NFATc2 vary depending on the cellular signaling landscape. Together with the finding that NFATC2 is indispensable for AML cell survival this study has elucidated novel roles(s) for NFATC2 in AML oncogenesis. Disclosures Massett: Kymab Ltd: Current Employment. Huang: Janssen Pharmaceutical Companies of Johnson & Johnson (China): Current Employment.

Leukaemic stem cell (LSC) persistence remains a major obstacle to curing chronic myeloid leukaemia (CML). The bone morphogenic protein (BMP) pathway is deregulated in CML, with altered expression and response to the BMP ligands shown to impact on LSC expansion and behaviour. In this study, we determined whether alterations in the BMP pathway gene signature had any predictive value for therapeutic response by profiling 60 CML samples at diagnosis from the UK SPIRIT2 trial and correlating the data to treatment response using the 18-month follow-up data. There was significant deregulation of several genes involved in the BMP pathway with ACV1C, INHBA, SMAD7, SNAIL1 and SMURF2 showing differential expression in relation to response. Therapeutic targeting of CML cells using BMP receptor inhibitors, in combination with tyrosine kinase inhibitor (TKI), indicate a synergistic mode of action. Furthermore, dual treatment resulted in altered cell cycle gene transcription and irreversible cell cycle arrest, along with increased apoptosis compared to single agents. Targeting CML CD34+ cells with BMP receptor inhibitors resulted in fewer cell divisions, reduced numbers of CD34+ cells and colony formation when compared to normal donor CD34+ cells, both in the presence and absence of BMP4. In an induced pluripotent stem cell (iPSC) model generated from CD34+ hematopoietic cells, we demonstrate altered cell cycle profiles and dynamics of ALK expression in CML-iPSCs in the presence and absence of BMP4 stimulation, when compared to normal iPSC. Moreover, dual targeting with TKI and BMP inhibitor prevented the self-renewal of CML-iPSC and increased meso-endodermal differentiation. These findings indicate that transformed stem cells may be more reliant on BMP signalling than normal stem cells. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to target LSC self-renewal and improve long-term outcome for patients.

Despite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) patients require life-long treatment due to leukemic stem cell (LSC) persistence. LSCs reside together with mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) in the bone marrow (BM) niche, which they modify to their advantage whilst impairing normal hematopoiesis. To date it has proved difficult to understand how LSCs both dominate and alter the niche, and to effectively target LSCs with current therapies. Recent studies have shown intrinsic and extrinsic deregulation of the bone morphogenetic protein (BMP) pathway in CML. These cells show altered stem cell fate, persistence, and response to BMP receptor (BMPR) antagonists, which affect cell behavior including cell cycle, apoptosis, and expansion (Laperrousaz et al. 2013; Grockowiak et al. 2017; Zylbersztejn et al. 2018; Toofan et al. 2018). We compared TKIs alone and in combination with a BMPR inhibitor to gain improved insights into BCR-ABL1 dependent and independent regulatory mechanisms within the niche environment using this therapeutic approach. K562 CML cell line and CML CD34+ primary cells were used in this study in combination with HS5 stromal cell co-culture. CML cells were treated with single or combination treatments of imatinib, the dual SRC-ABL1 TKI saracatinib, and the BMPR inhibitor dorsomorphin for 4h or 72h with and without BMP4 stimulation/co-culture. HSC and LSC interactions were also investigated using our artificial 3D BM niche model which comprises of magnetically levitated MSC spheroids embedded in medical-grade collagen type I, mimicking the BM biological and mechanical microenvironment, along with a high throughput microfluidic MSC spheroid formation system for drug testing. Kinase screens were performed on CML CD34+ cells using a chip-based microarray assay (PamGene) following 4h drug treatments. Phosphorylation data were then utilized for upstream kinase and pathway analysis using the metacore platform. This analysis facilitated identification of proteins showing a change in expression of ≥0.5-fold across all (n=3) patient samples, which was validated by qPCR and immunoblotting. Fluidigm multiplex qPCR was utilized to assess changes in expression of early response, self-renewal and differentiation genes. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays of primary CD34+CP-CML samples (n=3) following treatment. We demonstrated a synergistic mode of action upon inhibition of the BMP pathway in combination with TKI treatment, resulting in increased apoptosis (p< 0.001), altered cell cycle (G2-M, p< 0.01), fewer cell divisions, and a reduction in CD34+cells. Primary patient samples displayed differential gene expression in relation to response for genes involved in cell cycle (CDKN1A, CDKN2B, RB1; p< 0.05), self-renewal (PBX1; p< 0.01) and cell survival (GATA1, CKIT, p< 0.05). Kinase prediction analysis identified kinases involved in cell growth, development, differentiation, apoptosis, and cell-cell adhesion in treated sample lysates, with consequent pathway analysis highlighting transcription factors, ETS1, TP53 and C-MYC as main regulators across all common pathways. QPCR identified significant changes in expression of ETS1 (p< 0.001) and C-MYC (p< 0.01) following treatment. Within the top 10 deregulated pathways based on PamGene TK profiling we also identified GAB1 and GAB2 as common effectors, known to play an important role in growth and differentiation of myeloid cells, and which were demonstrated by immunoblotting to be considerably downregulated following 72h single and dual treatments in K562. Co-culture of CD34+ cells on stroma was chemo-protective, however dual treatment was still able to elicit strong anti-proliferative effects. We are currently investigating these promising findings in more detail using our 3D niche model and microfluidic spheroid platform. Taken together, these results provide vital insights into the mechanisms by which CML cells respond to current treatments, which is critical for improving therapeutic approaches and avoiding patient resistance or relapse. A combinatorial approach targeting the BMP pathway with BMP antagonists or small molecule inhibitors together with second-generation TKIs could open up new therapeutic possibilities. Disclosures No relevant conflicts of interest to declare.

Myeloid leukaemias share the common characteristics of being stem cell-derived clonal diseases, characterised by excessive proliferation of one or more myeloid lineage. Chronic myeloid leukaemia (CML) arises from a genetic alteration in a normal haemopoietic stem cell (HSC) giving rise to a leukaemic stem cell (LSC) within the bone marrow (BM) ‘niche’. CML is characterised by the presence of the oncogenic tyrosine kinase fusion protein breakpoint cluster region-abelson murine leukaemia viral oncogene homolog 1 (BCR-ABL), which is responsible for driving the disease through activation of downstream signal transduction pathways. Recent evidence from our group and others indicates that important regulatory networks involved in establishing primitive and definitive haemopoiesis during development are reactivated in myeloid leukaemia, giving rise to an LSC population with altered self-renewal and differentiation properties. In this review, we explore the role the bone morphogenic protein (BMP) signalling plays in stem cell pluripotency, developmental haemopoiesis, HSC maintenance and the implication of altered BMP signalling on LSC persistence in the BM niche. Overall, we emphasise how the BMP and Wnt pathways converge to alter the Cdx–Hox axis and the implications of this in the pathogenesis of myeloid malignancies.

The Hox family are master transcriptional regulators of developmental processes, including hematopoiesis. The Hox regulators, caudal homeobox factors (Cdx1-4), and Meis1, along with several individual Hox proteins, are implicated in stem cell expansion during embryonic development, with gene dosage playing a significant role in the overall function of the integrated Hox network. To investigate the role of this network in normal and aberrant, early hematopoiesis, we employed an in vitro embryonic stem cell differentiation system, which recapitulates mouse developmental hematopoiesis. Expression profiles of Hox, Pbx1, and Meis1 genes were quantified at distinct stages during the hematopoietic differentiation process and compared with the effects of expressing the leukemic oncogene Tel/PDGFRβ. During normal differentiation the Hoxa cluster, Pbx1 and Meis1 predominated, with a marked reduction in the majority of Hox genes (27/39) and Meis1 occurring during hematopoietic commitment. Only the posterior Hoxa cluster genes (a9, a10, a11, and a13) maintained or increased expression at the hematopoietic colony stage. Cdx4, Meis1, and a subset of Hox genes, including a7 and a9, were differentially expressed after short-term oncogenic (Tel/PDGFRβ) induction. Whereas Hoxa4-10, b1, b2, b4, and b9 were upregulated during oncogenic driven myelomonocytic differentiation. Heterodimers between Hoxa7/Hoxa9, Meis1, and Pbx have previously been implicated in regulating target genes involved in hematopoietic stem cell (HSC) expansion and leukemic progression. These results provide direct evidence that transcriptional flux through the Hox network occurs at very early stages during hematopoietic differentiation and validates embryonic stem cell models for gaining insights into the genetic regulation of normal and malignant hematopoiesis.

The complement system is a fundamental part of the body's innate immunity, providing a fast acting defense mechanism against invading organisms, tissue damage, or molecules identified as being ‘nonself.’ The classical complement cascade is initiated either through direct binding of the first component C1q to the pathogen surface or during the adaptive immune response whereby C1q binds to the antibody–antigen complexes. Following activation, pathogens are cleared through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The classical complement cascade therefore provides a key link between the effector mechanisms of innate and adaptive immunity. Advances in translational medicine have resulted in effective immunotherapies, particularly in the field of cancer, using monoclonal antibodies (MAbs). The MAbs bind to specific cell surface markers, thus initiating CDC through activation of the classical complement cascade, and ADCC. These immunotherapies highlight the potential to generate antitumor activities by harnessing the body's own natural immune response.

SHP1 and SHP2 tyrosine phosphatases have both been implicated in signalling pathways downstream of the interleukin-3 (IL-3) receptor. We have investigated the co-association of SHP1 and SHP2 with tyrosine-phosphorylated proteins in IL-3-dependent BaF/3 cells. We demonstrate that both SHP1 and SHP2 associate with Aic2A (beta chain of the IL-3 receptor), Gab2 and the paired inhibitory receptor B (PIR-B). The individual SH2 domains of SHP2 can independently bind Gab2, potentially important for the adapter function of SHP2. Association of both phosphatases with Aic2A and Gab2 increases upon IL-3 treatment. Recruitment of SHP1 to PIR-B also increases in response to IL-3, suggesting a functional link between inhibitory and cytokine receptor signalling. Aic2A is a rapid target for dephosphorylation following IL-3 stimulation and substrate-trapping versions of both phosphatases identify Aic2A and Gab2 as substrates for SHP1 and SHP2. These studies suggest that SH2-domain interactions are important for targetting these phosphatases to their substrates.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine, having different effects on primitive hemopoietic cells and terminally differentiated end-cells of the myeloid lineage. Human primitive hemopoietic cells (CD34+) were obtained from the peripheral blood after mobilization and induced to proliferate and then differentiate with a combination of cytokines in vitro. Cells at different time points were then used to analyze the expression of the GM-CSF receptor and GM-CSF mediated activation of the JAK 2-STAT 5 and MAP kinase pathways. Scatchard analysis as measured by radioligand binding revealed that freshly purified CD34+ cells expressed 36+/-1 high affinity receptors per cell (mean +/- SE, n = 3) and the level of expression was not significantly different after 3 days in culture, but rose five- to tenfold by day 8. The day 0 CD34+ cells were hyporesponsive to GM-CSF, but by 3 days in culture the cells were still morphologically immature but were actively proliferating and exhibited maximal GM-CSF induced JAK 2-STAT 5 and MAP kinase activation at the optimal time point. Further culture of the CD34+ cells resulted in myeloid differentiation associated with prolongation of MAP kinase activation but not JAK 2-STAT 5 activation. These data indicate that the JAK 2-STAT 5 and MAP kinase pathways are independently regulated and that changes in these signaling pathways occur with differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the proliferation and maturation of immature myeloid progenitor cells and primes mature cell function in phagocytes. To investigate whether the biochemical events following the binding of GM-CSF to its receptor are differentiation dependent we analysed GM-CSF mediated activation of the JAK 2-STAT 5 and MAP kinase pathways in undifferentiated HL-60 cells and HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO) or retinoic acid (RA). GM-CSF stimulated MAP kinase activation in both the undifferentiated and differentiated HL-60 cells. Activation of MAP kinase (expressed as a proportion of total cellular MAP kinase) was maximal at 5 min and of similar magnitude in both cell types. There was, however, a marked difference in the later kinetics of activation, with the response being transient in the undifferentiated cells and disappearing within 15 min, whereas it was prolonged and persisted for at least 60 min in the differentiated cells. GM-CSF mediated activation of STAT 5 was markedly increased (15–20-fold) after differentiation of HL-60 cells but the kinetics of activation did not change. The increase in STAT 5 activation was not due to a change in total cellular STAT 5 expression but correlated with increased JAK-2 protein levels. These data show that in the HL-60 cell model, differentiation modulates the activation of signalling molecules downstream of the GM-CSF receptor.

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated\ud using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein,\ud mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type\ud I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic\ud nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in\ud spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were\ud quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to\ud other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for\ud modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for\ud drug delivery studies.

The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed "minimal residual disease". Disease persistence suggests, that despite targeted therapeutic approaches, BCR-ABL-independent mechanisms exist which sustain the survival of a small population of cells, termed leukemic stem cells (LSC). We previously identified CD93 expression as a promising biomarker of LSC in chronic phase (CP)-CML. Our group has described the long term self-renewal potential of Lin-CD34+93+ CP-CML cells compared to their Lin-CD34+93- counterparts through LTCIC assays (n=3, p To interrogate this, we initially identified CP-CML subpopulations with the greatest functional capability compared to normal. Normal and CP-CML samples were FACS-sorted into HSC/LSC, CMP, GMP, and MEP sub-populations. Results suggest a significant change in functional status between normal and CP-CML subpopulations within the HSC/LSC compartment (lin-CD34+CD38-CD45RA-CD90+), where CML LSC demonstrated significantly increased proliferation (14 fold expansion; P90% of lin-CD34+CD38-CD45RA-CD90+ CML LSC from all patient samples were BCR-ABL positive. Subsequent experiments were confined to the LSC population. We hypothesized that lin-CD34+CD38-CD90+CD93- CML cells would have a more mature gene expression profile compared to lin-CD34+CD38-CD90+CD93+ cells. CP-CML cells were sorted into (1) lin-CD34+, (2) lin-CD34+CD38-CD90+CD93- and (3) lin-CD34+CD38-CD90+CD93+ populations. RNA was harvested at baseline from bulk populations (1) to (3) and cDNA was generated from single cells using the Fluidigm C1 autoprep system. Using Fluidigm technology, quantitative PCR of 90 lineage-specific and cell survival genes was performed within all populations of cells (1) to (3) in 'bulk' samples (n=3), and at single cell level (n=123 CD93+, n=120 CD93-single cells; n=3 samples in total). Bulk sample analysis demonstrated a significant increase in expression of lineage commitment genes within the lin-CD34+CD38-CD90+CD93- population, as shown by increased expression of GATA1 (p=0.0007), and CBX8 (p=0.0002). The lin-CD34+CD38-CD90+CD93+ population displayed a less lineage-restricted profile with increased expression of CDK6 (p=0.05), HOXA6 (ns), CDKN1C (ns) and CKIT (p=0.0014), compared to the lin-CD34+CD38-CD90+CD93- population. Furthermore, the two populations could be segregated by differential gene expression through gene clustering. At a single cell level, differences were noted in the frequency of expression between lin-CD34+CD38-CD90+CD93- and lin-CD34+CD38-CD90+CD93+ populations, particularly in GATA1, TPOR, and VWF. Although a statistically significant change was demonstrated in gene expression between the lin-CD34+CD38-CD90+CD93- and lin-CD34+CD38-CD90+CD93+ populations in a number of genes, we were not able to segregate the populations by differential expression using gene clustering. This highlights the heterogeneous nature of the cell populations and the inability to distinctly characterize between the two populations at a single cell level. Our results validate CD93 as a potential biomarker to separate the primitive CP-CML LSC population and highlight key lineage and cell survival pathways that are altered in CML LSC. The results demonstrate the heterogeneity seen within gene expression at the single cell level, which may allow for further insight into the CML LSC compartment with further analyses. Disclosures Wheadon: GlaxoSmithKline: Research Funding. Copland:Shire: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

Age is an independent risk factor in the outcome of acute myeloid leukemia (AML). Treatment of pediatric AML is often extrapolated from adult trials assuming disease homogeneity. However, age dependent clinical and molecular differences exist in AML and normal hematopoietic stem cells (HSCs) differ with age. We hypothesized that cellular age influences leukemogenesis. Using the NUP98HOXA9 (NH9) murine leukemia model, we addressed how intrinsic cellular age and the interplay with the bone marrow (BM) microenvironment impacts leukemic transformation. Lin-sca-1+cKit+ (LSK) cells isolated from fetal liver (FL) and BM at 3 week (w), 10w and 52w C57BL/6 mice were transduced with NH9 and in vitro transformation was assessed by colony forming cell (CFC) assays. NH9 transduced young (FL and 3w) and adult (10w and 52w) LSKs serially replated with a similar myeloid immunophenotype showing that LSKs from all ages transformed comparably in vitro in the absence of a BM niche. To investigate age related transformation in a BM microenvironment, NH9 transduced LSKs from all ages (at CFC2) were transplanted into 6w old C57BL/6 recipients. NH9 transduced LSKs from adult BM gave rise to leukemia with a shorter latency compared to young LSKs, with a reduction in median survival of almost 100 days. While adult LSKs resulted in a fully penetrant leukemia, only 50-75% of mice transplanted with young LSKs developed leukemia. A proportion of recipients transplanted with young LSKs developed acute lymphoblastic leukemia expressing lymphoid markers CD19 or B220 with an absence of myeloid markers. All mice transplanted with adult LSKs developed AML determined by morphology and flow cytometry, and immunohistochemistry of the leukemic blasts showed myeloid marker MPO expression. However, in AMLs generated from young LSKs, the blasts showed dual expression of MPO and the lymphoid marker CD3. Therefore, young LSKs retain expression of multi-lineage markers exhibiting lymphoid lineage potential. Together our data show that the presence of an adult microenvironment significantly alters the latency, penetrance and phenotype of the resultant leukemia from different aged stem cells. These effects may be cell intrinsic, or the BM microenvironment may be providing external cues, or may be as a result of the interaction between the leukemic cell and the BM niche cells. To address whether there may be cell intrinsic cues, targeted transcriptional profiling was assessed in NH9 transduced LSKs in vitro and in AMLs generated in vivo. Young LSKs transformed in vitro showed higher expression of genes associated with multiple hematopoietic lineages compared to adult LSKs, supporting the finding that young LSKs retain multi-lineage potential. In contrast, adult LSKs transformed in vitro showed higher expression of genes associated with a stem cell signature and upregulation of the bone morphogenetic protein (BMP) pathway. The BMP receptors BMPR1a, BMPR2, and the responsive SMAD1 were upregulated, and the antagonist Noggin was downregulated, suggesting that in adult transformed cells in vitro, the BMP pathway is primed for activation. The BMP pathway transcriptional targets were also perturbed, including upregulation of ID3 and downregulation of Creb3l1, which have been shown to have pro-oncogenic and tumor suppressive activities respectively. Significantly, upregulation of the BMP pathway was found in adult leukemic cells in the presence of a BM niche in vivo. Global transcriptional profiling by RNA-sequencing performed on in vivo generated AMLs from all 4 ages confirmed upregulation of the BMP pathway in adult AMLs. In further support of the role of the BM microenvironment in adult AML, pathways involved in cell adhesion and localization of the stem cell in the BM niche were enriched in adult AMLs. These data suggest a cell intrinsic role for the BMP pathway that may be exploited by transformed/leukemic cells in the presence of a BM niche. Our data may provide a mechanistic explanation for the enhanced myeloid leukemic potential of adult LSKs or, alternatively, the decreased susceptibility of young LSKs toward AML. Disclosures Wheadon: GlaxoSmithKline: Research Funding.

Primary cilia regulate hedgehog (Hh) signal transduction; these sensory organelles being present on most mammalian cells. Aberrant Hh activity has been implicated in malignancy with defective primary cilia expression linked to disease. The role the Hh pathway plays in AML has not been fully elucidated and it is unclear whether AML cells express primary cilia. Our aims were (1) to determine the presence of primary cilia and (2) further evaluate the role of Hh signalling in AML. Primary cilia were identified in all AML (n=23), and 20% of normal (n=10) bone marrow trephines (BMTs) by immunocytochemistry (ICC). Primary cilia were not identified in AML cell lines, primitive (n=4) or mature hematopoietic cells (n=6) isolated from peripheral blood, suggesting they are lost once cells migrate from the bone marrow (BM) microenvironment. Despite the heterogeneity of AML, analysis of 76 primary AML samples demonstrated clear evidence of Hh pathway activity with up regulation of SMO (p5-fold increase in SMO (n=28) showed a >5-fold up regulation of genes associated with chemoresistance and poor survival, including MECOM and FOXM1 (both p=0.0001), ABCC1, HOXA3, HOXA9, TWIST1 and SNAIL1 (all p The BM microenvironment is important in AML. Immunohistochemistry (IHC) on human AML BMTs (n=37) enabled analysis within this unique environment. Proteins associated with poor prognosis and a more aggressive phenotype were up regulated 2-30 fold, including BCL-2 (p In vitro, SMO inhibition with cyclopamine reduced cell proliferation in myelomonocytic cell lines (Kasumi-1 p This is the first report to demonstrate primary cilia on hematopoietic cells, with an increased frequency observed in AML. Their absence when cells migrate from the BM fits with their function and suggests a 'switching off' of the Hh pathway occurs on maturation. The ability of SMO inhibition to cause differentiation, in genetically diverse AML cell lines and primary AML is promising. SMO inhibition should continue to be explored as a potential therapy in AML. Disclosures Dobbin: Almac: Employment. Wheadon:GlaxoSmithKline: Research Funding. Copland:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Amgen: Honoraria; Shire: Honoraria.

The phenomenon of disease persistence in CP-CML under therapy with ABL kinase inhibitors suggests BCR-ABL-independent mechanisms are being exploited to sustain the survival of leukemic stem cells (LSC). Many self-renewal pathways have been implicated in their survival. The role of Notch signalling has yet to be fully elucidated, with conflicting reports suggesting a variety of roles in myeloid disease. The aims of this project were (1) to assess the expression and functional role of the Notch pathway in primary CD34+CP-CML cells, and (2) to assess differences in the functional role between stem and progenitor subpopulations in disease maintenance and progression. We hypothesized that Notch activation would have different functional effects dependent on disease phase and cell maturity. Extensive analysis of existing microarray datasets, GSE47927 and E-MTAB2481, comparing CP-CML LSCs to normal HSCs, showed that the Notch pathway was downregulated in CP-CML (p=0.05, both). To validate these results, using Fluidigm technology, mRNA expression of untreated CD34+ CP-CML cells (n=24) was compared to normal bone marrow CD34+ cells (n=5). The Notch pathway was silenced in untreated CP-CML, as evidenced by downregulation of Notch target genes, HES1 (p As CD34+ CP-CML cells abundantly express NOTCH2, we investigated whether the pathway could be reactivated through exogenous stimulation upon ligand binding. An OP9 co-culture system was utilized to allow for overexpression of Notch ligands, DLL1 or JAG1, using OP9GFP as control stroma. Compared to non-stromal conditions, culturing over 7 days on OP9GFP led to a significant increase in growth (p=0.02, n=5). The overexpression of JAG1 led to a further increase compared to OP9GFP (p=0.002). We confirmed that activation of the pathway was through JAG1, and not DLL1, by utilizing DAPT, where we observed a significant decrease in live cell counts within the OP9GFP (p=0.01) and OP9JAG1 (p=0.018) experimental arms. Upregulation of the pathway was confirmed with increased expression of HES1 at the gene level (n=4, p=0.001), andval1744 protein by IF (n=5). CP-CML samples (n=3) were FACS-sorted into Lin-CD34+38+ and Lin-CD34+38- populations to assess for functional variation in stem and progenitor populations. Following 7 days in culture, live cell counts showed a significant increase in Lin-CD34+38+ cells on OP9JAG1 co-culture (p=0.04). There was a decrease in cell growth of Lin-CD34+38- cells in the same experimental conditions (p=0.02), with an associated increased trend in late apoptotic cells (ns). We hypothesized that Notch activation would have a toxic influence on an immature CML LSC population. LTCIC assays showed a significant decrease in colonies for CD34+38-cells co-cultured with OP9JAG1 for 7 days (p In myeloid BP (n=4), in similar experiments, sorted Lin-CD34+38+ and Lin-CD34+38- populations did not demonstrate this functional profile of Notch activation, despite an upregulation in NOTCH2 receptor expression compared to normal (n=11) (p=0.0035). To investigate this further, we sorted CP (n=12), myeloid BP (n=11), and lymphoid BP (n=5) into stem and progenitor populations, before undertaking quantitative PCR of 90 self-renewal gene components of Wnt, Notch, Hedgehog, and BMP pathways. The self-renewal pathways were highly deregulated between CP and BP. The differential gene expression in lymphoid BP was comparable to myeloid BP. There was statistically significant upregulation in Wnt components in myeloid BP compared to CP, particularly TCF7(p=0.0011). We hypothesize that Wnt upregulation is preventing Notch activation in myeloid BP-CML. Taken together, our results identify that Notch activation may be a therapeutic approach to target CP-CML LSCs. However, this cannot be translated to myeloid BP, highlighting the complexity of self-renewal pathway interaction. Disclosures Wheadon: GlaxoSmithKline: Research Funding. Holyoake:Novartis: Honoraria, Research Funding; Bristol Myers Squib: Honoraria, Research Funding. Copland:ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Honoraria; Amgen: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The analysis of X-chromosome inactivation patterns has been used in a number of clinical situations such as the identification of carrier status in X-linked genetic disorders and the establishment of the monoclonal origin of tumors. Interpretation of the result obtained requires comparison with the constitutive pattern for the individual, and for hematopoietic malignancies, skin biopsies or cultured fibroblasts have often been used as the control tissue because normal cells of the same lineage as the malignancy are not generally available. However, this assumes that patterns in the different tissues are constitutionally the same. We have therefore compared X-chromosome inactivation patterns from peripheral blood (granulocytes, E- cells, and T cells), skin, and muscle from 20 hematologically normal females, and colonic mucosa from 9 individuals. In 11 patients (55%), the results obtained were similar for all tissues of an individual, but in 9 patients, significant differences were observed between tissues. The most consistent feature was a skewing in peripheral blood (> 75% expression of one allele) but not skin and/or muscle. These studies suggest that skin cannot be used as a control tissue for the interpretation of X-chromosome inactivation patterns in hematopoietic cells.

Wnt signaling plays several roles in hematopoiesis, promoting hemopoietic stem cell (HSC) self-renewal, providing proliferative signals for immature progenitors and regulating lineage commitment. To ascertain which Wnt proteins and receptors are important during hematopoietic development, we used two systems; in vitro hematopoietic differentiation of embryonic stem (ES) cells and tissues isolated from sites specific for hematopoiesis during mouse embryogenesis. Initially genes involved in hematopoiesis were profiled and indicate differentiating ES cells undergo a wave of primitive hematopoiesis (Day 3.75) similar to the mouse yolk sac, followed by a wave of more definitive hematopoiesis (Day 7.75) comparable to the aorta-gonad-mesonephros (AGM) and E15.5 liver with lineage commitment by Day 15. A similar biphasic expression pattern occurred for Wnt/Fzd/LRP genes with Wnt 3, 5a, 8a, Fzd4, and LRP5 becoming upregulated during primitive hematopoiesis, followed by Wnt3a, 6, 7b, 10b, and 16 during more definitive hematopoiesis. High expression of Wnt5a, Fzd4, and LRP5 during the first phase of hematopoiesis suggests these genes are involved in early hematopoietic regulation. Wnt3a and 16 were also expressed at specific stages, with Wnt16 detected when the earliest lymphoid progenitors are formed (AGM and 2 degrees BC of ES differentiation). Wnt3a expression corresponded with the induction of definitive hematopoiesis a period, which involves rapid expansion of HSC (Day 7.75 of ES differentiation, AGM and E15.5 liver). Supplementation with Wnt3a during ES hematopoietic differentiation increased proliferation and appeared to promote stem cell expansion. Overall this study provides valuable information on the Wnt/Fzd/LRP involved in supporting embryonic hematopoiesis.

The leukemic oncogene Tel/PDGFRbeta, was inducibly expressed in embryonic stem (ES) cells and the phenotypic and molecular changes occurring during hematopoietic differentiation investigated. Expression of Tel/PDGFRbeta resulted in an inability of ES cells to self-renew and caused a significant increase in myelopoiesis with a corresponding decrease in erythropoiesis. Analysis of gene expression patterns indicated a dramatic alteration in the levels of genes associated with self-renewal and differentiation, especially myelomonocytic genes in Tel/PDGFRbeta-expressing cells. This study indicates Tel/PDGFRbeta drives myelopoiesis by altering expression of genes involved in hematopoiesis and demonstrates the potential of this stem cell system to study oncogene-induced pathogenesis.

Polycythemia vera (PV) is characterized by the presence of the Janus kinase 2 (JAK2) V617F mutation in 97% of patients. This results in constitutive activation of the JAK/signal transduction and activators of transcription (STAT) pathway. JAK2 can also enter the nucleus directly following SUMOylation to effect alterations in histone function. Patients with PV display elevated peripheral blood counts, chronic inflammation and cytokine driven symptoms leading to morbidity and reduced quality of life. An important target of inflammation via JAK-mediated interferon (IFN) signalling is promyelocytic leukemia protein (PML), necessary for nuclear body (NB) formation. NBs act as a hub for transcriptional regulation and histone modification, bringing together multiple proteins to elicit diverse cellular functions including apoptosis, cellular senescence, DNA repair and inflammatory responses. Multiplex analysis of a 29 cytokine/chemokine panel in normal donor (n=8) and PV patient (n=10) serum, indicated significant upregulation of inflammatory cytokines through both JAK2-dependent and independent signalling, including IFNγ, interleukin (IL)-5, IL-12 (p< 0.05; JAK2 dependent), and CXCL10, MIP-1α and TNF-α (p< 0.001; JAK2 independent). PV patients also had significant deregulation of JAK2 dependent and IFN response genes (n=84 genes) especially upregulation of GBP1 (p< 0.05), MGST3 and SUMO3 (p< 0.001) and downregulation of PML (p< 0.01) and NFκB (p< 0.001). Using Duolink® in situ proximity ligation assays in both JAK2 V617F cell lines (UKE1 and SET2) and PV patient/normal donor monocytes, we demonstrate that JAK2 is SUMOylated and actively co-localises with PML in the cell nucleus. Importantly, there was a significant increase in the degree of interaction between JAK2 and PML in PV monocytes compared to normal donors (n=3, p< 0.001). Treatment of UKE1 and SET2 cell lines with the JAK1/2 inhibitor ruxolitinib led to a decrease in JAK2 dependent and IFN response genes as well as the degree of JAK2/PML co-localisation indicating active JAK2 mediates these responses. To determine whether the deregulation of these cellular processes is manifested by a functional alteration in the myeloid lineages of patients with PV, we analysed their monocyte profiles and the ability of monocytes to form M1 and M2 macrophages as well as neutrophil function. Monocytes exist in different subsets defined by the expression of CD14 and CD16. In PV, the proinflammatory intermediate subset was significantly increased (16.4% ±2.04 vs 6.5% ±1.08, p< 0.05, n=5) at the expense of the classical subset of monocytes (79% ±2.04 vs 88.9% ±1.07, p< 0.05, n=5). The non-classical, IL-1RA producing subset remains unchanged (3.6% ±0.38 vs 4.6% ±0.48 p= 0.1). Macrophages generated from these monocytes were polarized for M1 or M2 differentiation using GM-CSF and M-CSF respectively. They were then cultured in the presence of normal or PV patient serum with or without stimulation; IFNγ and lipopolysaccharide (LPS) for M1 macrophages and IL-4 for M2 macrophages. Culture supernatant was then assessed for the same 29 cytokine/chemokine panel as above and showed significant alterations including an increase in IL-6 and CXCL10 in PV derived M1 macrophages (p< 0.001), while M2 macrophages had a decrease in CXCL1 and increase in IL-1RA (p< 0.01). Neutrophils were cultured for 24 hours in the presence of normal or PV patient serum with or without stimulation with LPS and showed significant alteration in the production of several proinflammatory cytokines/chemokines compared to neutrophils cultured in Promocell Base Media DXF alone including IFNγ, VEGF, and CXCL10 (p< 0.001), indicating that serum from PV patients can elicit neutrophil activation. In conclusion, we have shown that inflammation is a major pathophysiological process in PV patients resulting in increased PML/JAK2 co-localisation, increased transcription of direct JAK2 target and IFN response genes, high circulating proinflammatory cytokine/chemokine levels, altered proinflammatory monocyte profiles, and significant change in the secretory production of mature granulocytes following stimulation. Treatment of JAK2 V617F positive cell lines with ruxolitinib significantly alters the inflammasome supporting its efficacy in the treatment of PV patients to alleviate the symptoms and complications of chronic inflammation. Disclosures Drummond: Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees. Copland:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The maintenance of murine embryonic stem (ES) cell self-renewal is regulated by leukemia inhibitory factor (LIF)-dependent activation of signal transducer and activator of transcription 3 (STAT3) and LIF-independent mechanisms including Nanog, BMP2/4, and Wnt signaling. Here we demonstrate a previously undescribed role for phosphoinositide 3-kinases (PI3Ks) in regulation of murine ES cell self-renewal. Treatment with the reversible PI3K inhibitor, LY294002, or more specific inhibition of class I(A) PI3K via regulated expression of dominant negative Deltap85, led to a reduction in the ability of LIF to maintain self-renewal, with cells concomitantly adopting a differentiated morphology. Inhibition of PI3Ks reduced basal and LIF-stimulated phosphorylation of PKB/Akt, GSK3alpha/beta, and S6 proteins. Importantly, LY294002 and Deltap85 expression had no effect on LIF-induced phosphorylation of STAT3 at Tyr(705), but did augment LIF-induced phosphorylation of ERKs in both short and long term incubations. Subsequently, we demonstrate that inhibition of MAP-Erk kinases (MEKs) reverses the effects of PI3K inhibition on self-renewal in a time- and dose-dependent manner, suggesting that the elevated ERK activity observed upon PI3K inhibition contributes to the functional response we observe. Surprisingly, upon long term inhibition of PI3Ks we observed a reduction in phosphorylation of beta-catenin, the target of GSK-3 action in the canonical Wnt pathway, although no consistent alterations in cytosolic levels of beta-catenin were observed, indicating this pathway is not playing a major role downstream of PI3Ks. Our studies support a role for PI3Ks in regulation of self-renewal and increase our understanding of the molecular signaling components involved in regulation of stem cell fate.

The cytosolic SHP-2 (Src homology protein tyrosine phosphatase 2) has previously been implicated in IL-3 (interleukin-3) signalling [Bone, Dechert, Jirik, Schrader and Welham (1997) J. Biol. Chem. 272, 14470 –14476; Craddock and Welham (1997) J. Biol. Chem. 272, 29281–29289; Welham, Dechert, Leslie, Jirik and Schrader (1994) J. Biol. Chem. 269, 23764–23768; Qu, Nguyen, Chen and Feng (2001) Blood 97, 911–914]. To investigate the role of SHP-2 in IL-3 signalling in greater detail, we have inducibly expressed WT (wild-type) or two potentially substrate-trapping mutant forms of SHP-2, generated by mutation of Asp-425 to Ala (D425A) or Cyst-459 to Ser (C459S), in IL-3-dependent BaF/3 cells. Effects on IL-3-induced tyrosine phosphorylation, signal transduction and functional responses were examined. Expression of C459S SHP-2 protected the β-chain of the murine IL-3R (IL-3 receptor), the adaptor protein Gab2 (Grb2-associated binder 2), and a cytosolic protein of 48 kDa from tyrosine dephosphorylation, consistent with them being bona fide substrates of SHP-2 in IL-3 signalling. The tyrosine phosphorylation of a 135 kDa transmembrane protein was also protected upon expression of C459S SHP-2. We have identified the inhibitory immunoreceptor PECAM-1 (platelet endothelial cell adhesion molecule-1)/CD31 (cluster determinant 31) as a component of this 135 kDa substrate and also show that IL-3 can induce tyrosine phosphorylation of PECAM-1. Expression of WT, C459S and D425A forms of SHP-2 had little effect on IL-3-driven proliferation or STAT5 (signal transduction and activators of transcription) phosphorylation or activation of protein kinase B. However, expression of WT SHP-2 increased ERK (extracellular-signal-regulated kinase) activation. Interestingly, expression of C459S SHP-2 decreased ERK activation at later times after IL-3 stimulation, but potentiated IL-3-induced activation of Jun N-terminal kinases. In addition, expression of C459S SHP-2 decreased cell survival in suboptimal IL-3 and upon IL-3 withdrawal. These findings indicate that SHP-2 plays an important role in mediating the anti-apoptotic effect of IL-3 and raises the possibility that PECAM-1 participates in the modulation of cytokine-induced signals.

The TEL/PDGFβR oncogenic fusion protein is the product of the t(5;12)(q33; p13) translocation recurrently found in patients with chronic myelomonocytic leukemia (CMML). To investigate the coupling of molecular signaling events activated by TEL/PDGFβR to functional responses, we expressed TEL/PDGFβR in interleukin 3 (IL-3)–dependent BaF/3 cells using the tetracycline-regulated expression system. Induction of TEL/PDGFβR expression led to increased cell survival following IL-3 withdrawal and constitutive activation of protein kinase B (PKB), signal transducer and activator of transcription 5 (STAT5), extracellular signal-regulated kinases 1/2 (ERK1/2), Jun N-terminal kinases 1/2 (JNK1/2), and p38 mitogenactivated protein kinase (MAPK) pathways. However, inducible expression of TEL/PDGFβR failed to generate factor-independent cells, whereas constitutive expression of TEL/PDGFβR did, albeit at low frequency, suggesting the duration of TEL/PDGFβR expression is important for transformation. Surprisingly, in cells induced to express TEL/PDGFβR, IL-3–dependent growth was dramatically reduced as a result of increased apoptosis of cells receiving combined IL-3 and TEL/PDGFβR signals. We demonstrate that TEL/PDGFβR expression augmented IL-3–induced activation of PKB, STAT5, ERK1/2, p38, and JNK1/2. Inhibition of neither phosphoinositide-3 kinases nor p38 MAPKs reduced the inhibition of IL-3–driven proliferation observed when TEL/PDGFβR was expressed. However, inhibition of MEKs or JNKs partially reversed the combined inhibitory effects of TEL/PDGFβR and IL-3 on proliferation and survival. These results suggest that the combination of TEL/PDGFβR and IL-3–induced signals activate apoptosis through ERK and JNK MAPK-dependent pathways. Given that in vivo hematopoietic cells are in contact with a variety of cytokines, our results have important implications for cellular responses in the pathogenesis of CMML.

The acquisition of cytogenetic abnormalities and genetic mutations contribute towards the heterogeneity of CLL progression. Patients harbouring NOTCH1 mutations (NOTCH1MUT) are identified as a poor prognostic subset, with the mutation conferring increased cell survival and proliferation. A retrospective study of the CLL8 clinical trial identified that NOTCH1MUT patients did not benefit from the addition of rituximab (RTX) to standard first line therapy. While trial results investigating the efficacy of combining ofatumumab (OFA) and chlorambucil suggested an initial benefit for both NOTCH1 wild type (NOTCH1WT) and NOTCH1MUT patients, progression free survival of NOTCH1MUT patients was not significantly enhanced by the inclusion of OFA. This study prompted us to analyse how NOTCH1 mutations impact on OFA/RTX-mediated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We also assessed how proliferating CLL cells respond to OFA in NOTCH1WT and NOTCH1MUTsamples at the level of CD20 expression, ability to induce CDC, and changes in gene expression profiles (GEPs). Of 89 CLL patients screened for NOTCH1 mutations, 8 had NOTCH1 mutations in the absence of 11q and 17p deletions. We assessed the ability of these cells to undergo ADCC and CDC following OFA or RTX treatment in vitro, and detected no significant difference in response between NOTCH1MUT and NOTCH1WTCLL patients. However in both CLL cohorts we did demonstrate that OFA more efficiently induced both ADCC and CDC compared with RTX. Interaction of CLL cells with the microenvironment of lymphoid organs promotes survival and proliferation, and generates cytoprotective signals that must be overcome to provide a curative therapy for CLL. To compare how NOTCH1MUT and NOTCH1WT CLL patients respond to OFA treatment in vitro in pro-proliferative conditions, three different culture systems were used: plastic (media alone), NTL (stromal cells) and NTL-CD154/IL-4 (stromal cells expressing CD40L supplemented with IL-4). Flow cytometric analyses revealed differential regulation of CD20 expression levels between NOTCH1MUT and NOTCH1WT samples, with 40% of NOTCH1WT CLL samples up-regulating CD20 surface expression on NTL-CD154/IL-4, with no change observed in NOTCH1MUT samples. Microenvironmental conditions, in the presence/absence of OFA impacted the OFA-mediated CDC response between NOTCH1WT and NOTCH1MUT patients. Indeed, post NTL-CD154/IL-4 co-culture the OFA-CDC response was significantly higher in NOTCH1WT samples, with a mean increase in CDC of 36.5% (p=0.005; n=5), compared to CLL cells on plastic, while an elevation in CDC did not occur in NOTCH1MUT CLL patients. Conversely when NOTCH1MUTpatients were pre- incubated with OFA on NTL-CD154/IL-4 they produced significantly higher levels of CDC (37.5%; p=0.02; n=5) when compared to pre-incubation with OFA on plastic. GEPs of fresh NOTCH1WT and NOTCH1MUT CLL samples revealed that NOTCH1MUT samples express significantly higher levels of DELTEX and lower levels of HES5, TP73, RRAD and CCNE2. Co-culture of both CLL cohorts on NTL-CD154/IL-4 resulted in a decrease in NOTCH1 levels, and other components of the Notch signalling pathway - HES1, LFNG and ADAM10. While both CLL cohorts up-regulated ROR2, FOS and JUN expression upon NTL-CD154/IL-4 co-culture, this was higher in NOTCH1MUT CLL cells. Interestingly, ROR2, FOS and JUN have been linked with Ca2+ channel activity and Ca2+ signalling, together with DAPK1 and RRAD, which were also deregulated in NOTCH1MUT CLL samples. In conclusion, our studies indicate that the OFA-CDC response to microenvironmental stimulation cannot be entirely attributed to CD20 expression levels. Reduced OFA-CDC activity in NOTCH1MUT samples may be due to deregulation of genes involved in Ca2+ signalling, highlighting the role of CD20 as a store-operated Ca2+ channel. Disclosures Middleton: GlaxoSmithKline UK Ltd: Research Funding. Leach:GlaxoSmithKline UK Ltd: Research Funding. Michie:GlaxoSmithKline UK Ltd: Research Funding. Wheadon:GlaxoSmithKline UK Ltd: Research Funding.

The generation of hematopoietic stem cells (HSCs) during development is a complex process linked to morphogenic\ud signals. Understanding this process is important for regenerative medicine applications that require in vitro production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic\ud differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates\ud that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst\ud maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/\ud Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn\ud lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription\ud factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling\ud also augmented early hematopoietic and multipotent progenitor (MPP) formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population,with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin\ud signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.

A fundamental property of hematopoietic stem cells (HSCs) is the ability to self-renew. This is a complex process involving multiple signal transduction cascades which control the fine balance between self-renewal and differentiation through transcriptional networks. Key activators/regulators of self-renewal include chemokines, cytokines and morphogens which are expressed in the bone marrow niche, either in a paracrine or autocrine fashion, and modulate stem cell behaviour. Increasing evidence suggests that the downstream signaling pathways induced by these ligands converge at multiple levels providing a degree of redundancy in steady state hematopoiesis. Here we will focus on how these pathways cross-talk to regulate HSC self-renewal highlighting potential therapeutic windows which could be targeted to prevent leukemic stem cell self-renewal in myeloid malignancies.

Objective Fusion genes involving the platelet-derived growth factor receptor-β ( PDGFRβ ) are found in a subgroup of myeloproliferative neoplasms, with one such fusion, Tel/PDGFRβ found in a subset of chronic myelomonocytic leukemia patients. Tel/PDGFRβ results in constitutive activation of several signaling pathways and induces a myeloproliferative disease in mice, with signals via tyrosines 579/581 identified as being important for this phenotype. In this study, we have used a tetracycline-regulated system to express wild-type and the mutated F2 Tel/PDGFRβ to identify the key signaling pathways, which drive Tel/PDGFRβ-induced differentiation of embryonic stem (ES) cells. Materials and Methods The leukemic oncogene Tel/PDGFRβ and Tel/PDGFRβ-F2 were inducibly expressed in ES cells and their effects on self-renewal, signal transduction, and gene expression patterns analyzed. Results Tel/PDGFRβ activated several major signal transduction pathways (signal transducers and activators of transcription [STAT] 3, STAT5, mitogen-activated protein kinases, phosphatidylinositol-3 kinase) in ES cells, but only specific inhibition of the mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) or STAT5 pathways was able to significantly prevent Tel/PDGFRβ-induced differentiation and restore ES-cell self-renewal. Inhibiting the tyrosine kinase activity of the oncogene using Gleevec or PDGFRβ inhibitor III also substantially prevented Tel/PDGFRβ-induced differentiation and its ability to upregulate key genes involved in myelopoiesis. Tyrosines 579/581 played a critical role in mediating signals via the Ras/ERK and STAT5 pathways, with dual targeting of the tyrosine kinase activity of Tel/PDGFRβ and the MEK/ERK pathway completely preventing Tel/PDGFRβ-induced differentiation. Conclusion These findings suggest that targeted disruption of key signaling pathways in combination with the tyrosine kinase activity of leukemic oncogenes, such as Tel/PDGFRβ, may result in more efficacious therapies for suppressing leukemic progression in the clinical setting.