Salt inducible kinases 2 and 3 are required for thymic T cell development

Salt Inducible Kinases (SIKs), of which there are 3 isoforms, are established to play roles in innate immunity, metabolic control and neuronal function, but their role in adaptive immunity is unknown. To address this gap, we used a combination of SIK knockout and kinase-inactive knock-in mice. The combined loss of SIK1 and SIK2 activity did not block T cell development. Conditional knockout of SIK3 in haemopoietic cells, driven by a Vav-iCre transgene, resulted in a moderate reduction in the numbers of peripheral T cells, but normal B cell numbers. Constitutive knockout of SIK2 combined with conditional knockout of SIK3 in the haemopoietic cells resulted in a severe reduction in peripheral T cells without reducing B cell number. A similar effect was seen when SIK3 deletion was driven via CD4-Cre transgene to delete at the DP stage of T cell development. Analysis of the SIK2/3 Vav-iCre mice showed that thymocyte number was greatly reduced, but development was not blocked completely as indicated by the presence of low numbers CD4 and CD8 single positive cells. SIK2 and SIK3 were not required for rearrangement of the TCRβ locus, or for low level cell surface expression of the TCR complex on the surface of CD4/CD8 double positive thymocytes. In the absence of both SIK2 and SIK3, progression to mature single positive cells was greatly reduced, suggesting a defect in negative and/or positive selection in the thymus. In agreement with an effect on negative selection, increased apoptosis was seen in thymic TCRbeta high/CD5 positive cells from SIK2/3 knockout mice. Together, these results show an important role for SIK2 and SIK3 in thymic T cell development.

Haematopoietic thrombocyte precursors in rat femoral and sternal bone marrow

This research presents the first findings on thrombopoiesis for Wistar rats. Haemopoietic cells from the femur and the sternum were analysed by light microscopy in combination with infrared and near-ultraviolet light for fine cytoplasmic structure analysis. Five main types of thrombocyte precursor cells were identified in the bone marrow samples: megakaryoblast, promegakaryocyte and megakaryocyte (basophilic, acidophilic and thrombocytogenic). More intensive thrombopoiesis and morphologically differentiated cells were found in sternum samples.

Histogenesis of Human Fetal Spleen

Background and Aims: Spleen is the largest secondary lymphatic organ. It acts as a graveyard for RBCs, is essential for immune responses, performs lymphopoiesis in adults and haemopoiesis in fetuses. The present study was conducted to assess the histogenesis of spleen in human fetuses in view of existing literature. Material and Methods: The study was carried out on 34 formalin preserved human fetuses procured from Dr Sushila Tiwari Government Hospital, Haldwani with due clearance from ethical committee. The 6 pm sections of the spleen were stained with Haematoxylin and Eosin and observed under light microscope. Results: At 14 tol5 weeks, spleen had extensive sinusoids filled with RBCs and few lymphocytes. At 16-18 weeks, trabecular arteries were noticed more towards centre along with extensive haemopoietic cells in the venous sinusoids. By 20th week lymphocytic aggregation had started around arterioles. By 24 weeks periarteriolar lymphatic sheath was clearly observed. At term (37-40 weeks), classical primary lymphoid follicle was present but germinal centers were not observed. Conclusion: During earlier differentiation, spleen symbolizes the function of haemopoietic activities and gradually during subsequent gestation; it establishes its identity as a principle lymphoid tissue.

Interaction of c-Myb with p300 Is Required for the Induction of Acute Myeloid Leukemia by Human AML Oncogenes, and Represents a Potential Therapeutic Target.

Abstract 2402 The MYB oncogene is widely expressed in acute leukaemias and is important for the continued proliferation of leukemia cells, raising the possibility that MYB may be a therapeutic target. However realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal haemopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for Myb's intrinsic transforming activity (1). Here we use haemopoietic cells from the Booreana mouse strain, which carries a mutation in Myb that prevents interaction with CBP/p300 (see Fig. 1 and ref. 2), to examine the requirement for this interaction in myeloid transformation and leukaemogenesis. Using this strain and a strain (plt6) carrying a “complementary” mutation in p300 (3), we show that the Myb-p300 interaction is essential for in vitro transformation by the myeloid leukaemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type (WT) mice, Booreana cells fail to induce leukaemia upon transplantation into irradiated recipients following transduction with an AML1-ETO9a retrovirus (Fig. 2). These data, as well as reinforcing the notion that MYB is an essential cofactor for myeloid leukaemogenesis, highlight disruption of the Myb-p300 interaction as a potential therapeutic strategy for AML. Furthermore, our results suggest that such a strategy would have a useable therapeutic index since Booreana mice, unlike Myb null mice, are viable. We have started to explore the use of small peptides to provide proof-of-principle for this approach. We have also begun to explore the molecular basis of the requirement for MYB, and the Myb-p300 interaction, in AML using gene expression profiling. This has highlighted several Myb target genes – identified by our previous ChIP-Seq analysis (4) - that have been previously implicated in myeloid leukaemogenesis, as being differentially expressed between WT and Booreana cells transduced with AML1-ETO9a. One striking example is the apparent Myb-p300 dependent activation of Ets2 and Gfi1, accompanied by repression of Gata2 and Gfi1b; this pattern has previously been reported in MLL-AF9 and MOZ-TIF-induced murine AML (5). Disclosures: No relevant conflicts of interest to declare.

Haemopoietic System of the Anurans: The Role of Bone Marrow and Liver

Haemopoietic System of the Anurans: The Role of Bone Marrow and Liver The haemopoietic activity of the frog, Pelophylax ridibundus was investigated during the year. Liver and bone marrow myelograms were examined in the different seasons using the special indexes and coefficients. It was shown the presence of the erythroid and granulocytic differentiation during the year in the both organs. In the bone marrow is changing the total number of the non-diferentiated haemopoietic cells, but ratio between erythroid and granulocytic progenitors is stabile. The haemopoietic activity of the liver has more prominent season variation in comparison with the marrow, but their total significance is comparable. The erythrocytic differentiation is more prominent during the summer and autumn, but granulocytic one took place in the spring and summer.

Investigating the Effect of Hsp90 Inhibitors on Initiating Cells in Childhood Acute Lymphoblastic Leukaemia

Abstract 2952 Enhanced risk stratification protocols and intensified therapies have improved outcomes and reduced the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). Nevertheless, 20% of patients relapse, due to failure to eradicate the disease. Current chemotherapeutic regimens are designed around the properties of the bulk leukaemia cells, which might differ from those of the leukaemia initiating cell populations (LIC). Therefore, if drugs have no effect on LIC, these cells may expand and eventually cause relapse. Since several populations in childhood ALL have been shown to have LIC properties, developing therapies that are effective against all LIC populations may prevent relapse. We have previously shown that the NF-κB inhibitor parthenolide (PTL) could prevent proliferation and engraftment of unsorted cells and all LIC populations in NSG mice, in most cases examined. Heat shock protein (Hsp) 90 inhibitors are promising targets in cancer therapy. Targeting this protein has a combined impact on many oncogenic pathways involved in malignant progression. In order to investigate whether the ablation of LIC that we observed using PTL could be improved, we examined the effects of two Hsp90 inhibitors on childhood ALL cells in this study. 17-DMAG is used in preclinical and clinical phase I and II testing in breast cancers. It targets the binding site of ATP in Hsp90 and has low toxicity and high oral bioavailability. Celastrol is a novel Hsp90 inhibitor, which has recently been shown to eradicate LIC in AML by inhibiting NF-κB survival signals and inducing oxidative stress. Dose-response effects using 0.01–100nM of each Hsp90 inhibitor were assessed in primary T- and B-ALL cases and on normal haemopoietic cells at 24, 48 and 72h. Cells were stained with Annexin V and PI then viability and apoptosis were assessed by flow cytometry. An IC50 was observed in leukaemia samples using 1nM of both 17-DMAG and Celastrol after 72h. Increasing the dose of 17-DMAG to 100nM and reducing the incubation time to 48 hours for each drug further reduced ALL cell survival, without impacting normal cells. At these doses, 17-DMAG reduced the viability of T-ALL cells to 36±30% and B-ALL cells to 32±13%. In T-ALL cases, 43±15% survived treatment with Celastrol and similar results were observed with B-ALL cells (43±16%). Normal haemopoietic cells were relatively unaffected at these doses (17-DMAG: 81±2% and Celastrol: 86±36%). Subsequently, T-ALL cells were sorted for expression of CD34 and CD7 and B-ALL cells were sorted for CD34 and CD19. The subpopulations were treated with 1nM of each inhibitor and the results compared to those observed using untreated controls. However, at these concentrations the drugs had limited effects on the ALL subpopulations; 31–100% and 28–89% T-ALL subpopulations survived treatment with 17-DMAG and Celastrol respectively. Similar results were observed with B-ALL subpopulations, 9–86% survived treatment with 17-DMAG and 62–100% survived following Celastrol treatment. A number of studies have shown that a regulatory loop may exist between Hsp90 and NF-κB in that downregulation of NF-κB could lead to reduction in Hsp90 protein levels. Therefore, we investigated whether using the Hsp90 inhibitors in combination with a NF-κB inhibitor would be more effective. Samples were treated with 100nM 17-DMAG or 1nM Celastrol for 48h and 7.5mM PTL was added for the last 24 hours. In 3 T-ALL cases, PTL reduced the viability to 28±13%, 17-DMAG to 25±12% and Celastrol to 35±15%. When PTL was used in combination, cell survival was further reduced to only 18±9% (PTL + 17-DMAG) and to 19±10% (PTL + Celastrol). In 3 B-ALL cases, PTL treatment reduced viability to 57±9%, similar results were seen with 17-DMAG (59±6%), while Celastrol appeared to be the most effective of the 3 drugs (38±4%, P=0.02). When PTL was combined with the Hsp90 inhibitors the cell killing was increased by 2 fold compared to PTL or 17-DMAG alone (PTL + 17-DMAG: 31±6%, P=0.04 and PTL + Celastrol: 28±3% P=0.01). Results to date indicate a promising role for the use of Hsp90 inhibitors with PTL and data on the functional ability of treated LIC populations will provide further information on the potential of these drug combinations. In conclusion, these Hsp90 inhibitors were as effective as PTL against childhood leukaemia cells and when used in combination with PTL, cell survival was further reduced by up to 20%. Disclosures: No relevant conflicts of interest to declare.

Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

Cytokines and their receptors regulate haemopoiesis by controlling cellular growth, survival and differentiation. Thus it is not surprising that mutations of cytokine receptors contribute to the formation of haemopoietic disorders, including cancer. We recently identified transforming properties of IL27R, the ligand-binding component of the receptor for interleukin-27. Although wild-type IL27R exhibits transforming properties in haemopoietic cells, in the present study we set out to determine if the transforming activity of IL27R could be enhanced by mutation. We identified three mutations of IL27R that enhance its transforming activity. One of these mutations is a phenylalanine to cysteine mutation at residue 523 (F523C) in the transmembrane domain of the receptor. The two other mutations identified involve deletions of amino acids in the cytoplasmic juxtamembrane region of the receptor. Expression of each of these mutant IL27R proteins led to rapid cytokine-independent transformation in haemopoietic cells. Moreover, the rate of transformation induced by these mutants was significantly greater than that induced by wild-type IL27R. Expression of these IL27R mutants also induced enhanced activation of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling compared with wild-type. An activating deletion mutation of IL27R enhanced homodimerization of the receptor by a mechanism that may involve disulfide bonding. These transforming IL27R mutants displayed equal or greater transforming activity than bona fide haemopoietic oncogenes such as BCR–ABL (breakpoint cluster region–Abelson murine leukaemia viral oncogene homologue) and JAK2-V617F. Since IL27R is expressed on haemopoietic stem cells, lymphoid cells and myeloid cells, including acute myeloid leukaemia blast cells, mutation of this receptor has the potential to contribute to a variety of haemopoietic neoplasms.

ER stress modulates cellular metabolism

Changes in metabolic processes play a critical role in the survival or death of cells subjected to various stresses. In the present study, we have investigated the effects of ER (endoplasmic reticulum) stress on cellular metabolism. A major difficulty in studying metabolic responses to ER stress is that ER stress normally leads to apoptosis and metabolic changes observed in dying cells may be misleading. Therefore we have used IL-3 (interleukin 3)-dependent Bak−/−Bax−/− haemopoietic cells which do not die in the presence of the ER-stress-inducing drug tunicamycin. Tunicamycin-treated Bak−/−Bax−/− cells remain viable, but cease growth, arresting in G1-phase and undergoing autophagy in the absence of apoptosis. In these cells, we used NMR-based SIRM (stable isotope-resolved metabolomics) to determine the metabolic effects of tunicamycin. Glucose was found to be the major carbon source for energy production and anabolic metabolism. Following tunicamycin exposure, glucose uptake and lactate production are greatly reduced. Decreased 13C labelling in several cellular metabolites suggests that mitochondrial function in cells undergoing ER stress is compromised. Consistent with this, mitochondrial membrane potential, oxygen consumption and cellular ATP levels are much lower compared with untreated cells. Importantly, the effects of tunicamycin on cellular metabolic processes may be related to a reduction in cell-surface GLUT1 (glucose transporter 1) levels which, in turn, may reflect decreased Akt signalling. These results suggest that ER stress exerts profound effects on several central metabolic processes which may help to explain cell death arising from ER stress in normal cells.