Inhibition Of Erythropoiesis By Iron Overload Is Mediated Through TGFβ Signaling

Abstract Several diseases, such as β-thalassemia and myelodysplastic syndrome, are characterized by iron overload and dysfunction erythropoiesis. Multiple retrospective studies suggest that iron overload is associated with increased disease burden and shortened survival in patients. It is general consensus that iron overload has an inhibitory effect on erythroid differentiation, but direct evidence is lacking and the mechanisms underlying this phenomenon are incompletely understood. Excess iron is associated with the generation of reactive oxygen species (ROS) that result in cellular damage. We hypothesized that iron overload leads to the stimulation of myelosuppressive pathways that result in decreased erythropoiesis and resulting cytopenias. We first evaluated the effect of exogenous iron on the growth of primary human CD34+ stem cells and demonstrated that iron overload leads to decreased erythroid colony formation in vitro. Furthermore, in our in vivo experiments, we administered a total of 130 mg iron dextran IP over 20 days to C57BL/6 mice and compared these mice with PBS injected age and gender matched controls (n=5/group). We demonstrate that iron injection results in fewer circulating reticulocytes (200 vs. 310 x 109 cells/L; P=0.001) and hemoglobin (13.8 vs. 15 g/dL; P=0.0004). Using anti-CD44 and TER119 as flow cytometric markers, our data reveals a reduction in bone marrow erythroid burden (total erythroid precursors 9.6 vs. 15.3% in controls; P=0.04) with a disproportionately greater effect on orthochromatophilic erythroblasts (4.4 vs. 6.9%; P=0.02). As expected, bone marrow-derived erythroid precursors from iron loaded mice have more ROS (MFI 1497 vs. 958; P=0.05) as measured by flow cytometry, again with a disproportionately greater effect on orthochromatophilic erythroblasts (MFI 671 vs. 403; P=0.01). No effect on cell cycle was observed. To evaluate the mechanism behind iron induced suppression of erythropoiesis, we conducted a functional screen with variety of cytokine inhibitors. We observed that a specific inhibitor of the TGFβ receptor I kinase led to reversal of iron induced suppression of erythroid colonies from primary CD34+ cells in vitro. The ability of TGFβ kinase inhibitor, LY-215, in reversing iron mediated suppression was confirmed in variety of hematopoietic cell lines, resulting in the inhibition of iron induced apoptosis in these cells. Taken together, our data demonstrates for the first time that iron overload has direct suppressive effects on erythropoiesis. These effects are reversed by specific inhibitors of the TGFβ receptor I kinase and thus provide a preclinical rationale for these inhibitors in anemias associated with iron overload. Disclosures: No relevant conflicts of interest to declare.

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Enhancing Functional Platelet Release In Vivo from in Vitro-Grown Megakaryocytes Using the Protein Kinase Inhibitor SU6656

Blood ◽

10.1182/blood.v128.22.1029.1029 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1029-1029

Author(s):

Danuta Jadwiga Jarocha ◽

Karen K Vo ◽

Randolph B Lyde ◽

Vincent M Hayes ◽

Mortimer Poncz

Keyword(s):

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Specific Inhibitor ◽

Annexin V ◽

Rock Inhibitor ◽

Medicine Research ◽

Nsg Mice ◽

Platelet Release

Abstract The clinical demand for platelet transfusions is increasing, threatening the ability to obtain sufficient healthy donors to provide these platelets. Advances in regenerative medicine research have opened the possibility of generating sufficient in vitro-grown megakaryocytes and consequent platelets to supply a portion of the clinical platelet transfusion demand. We have shown that infusing megakaryocytes for obtaining released, functional platelets is a viable alternative strategy than trying to release platelets in vitro. However, for both approaches, in vitro-cultured megakaryocytes have lower ploidy and release fewer platelets than likely occurs in vivo by primary cells. SU6656 inhibitor, a Src kinase inhibitor, has been shown to influence ploidization in several megakaryocyte-like line with purported increase in proplatelets release. However, in our hands, other agents - such as the ROCK inhibitor Y27632 - while increasing polyploidization markedly, inhibited platelet release per infused megakaryocyte in vivo. We grew megakaryocytes from CD34+ cells for 12 days with or without SU6656 (2.5 µM) supplementation during the last 4 days. We found that the SU6656 inhibitor only increased the number of CD34+-derived megakaryocytes by ~15% at the end of the 12 day growth, but more markedly increase the percent of large megakaryocytes measured by FSC parameter in flow cytometry evaluation from 28 up to 41% and percent of high granular megakaryocytes from 27 to 45%. These changes were accompanied with a shift in average ploidy from 4.9 to 6.9 (p<0.0003, N=6). Notably, SU6656-treated megakaryocytes released ~4-fold more platelets per infused megakaryocytes in immunocompromized NSG mice than untreated similarly in vitro-grown megakaryocytes. By 24 hrs, there were 6.5-fold platelets from the infused SU6656-treated megakaryocytes than control untreated (p<0.037, N=6). Released platelets from the drug-treated and untreated megakaryocytes had similar levels of percent thiazole orange positivity as an indication that they were young platelets. Importantly, baseline annexin V, CD62p and PAC1 binding prior to agonist exposure were also similarly and increased to the same extent after thrombin (1U/ml) stimulation. Additionally, incorporation into a growing cremaster laser injury-induced thrombus in vivo was similar further indicating retained function by the platelets released from the drug-treated megakaryocytes. A number of strategies such as modifying the level of transcription factors have been proposed to increase the size, ploidy or proplatelets release from in vitro-grown megakaryocytes. In none of these cases have these released platelets in vivo biology been examined and demonstrated to replicate high release number per megakaryocyte and retained functionality. We show that terminal exposure of in vitro-grown megakaryocytes to the non-specific inhibitor SU6656 significantly increases in vivo yield while leaving in vivo half-life and functionality intact. The exact pathway affected by SU6656 that leads to these results is now being pursued. Disclosures No relevant conflicts of interest to declare.

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SD-208, a Novel Transforming Growth Factor Beta Receptor I Kinase Inhibitor, Can Stimulate Hematopoiesis in Myelodysplastic Syndrome Progenitors.

Blood ◽

10.1182/blood.v106.11.3448.3448 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3448-3448

Author(s):

Amit Verma ◽

Tony A. Navas ◽

Jing Ying ◽

Aaron N. Nguyen ◽

Perry Pahanish ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Transforming Growth Factor Beta ◽

Mononuclear Cells ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

Specific Inhibitor ◽

Low Grade ◽

Bone Marrow Biopsies

Abstract Transforming Growth Factor β (TGF-β) is a myelosuppressive cytokine that has been implicated in the ineffective hematopoiesis seen in myelodysplastic syndromes (MDS). Overactivation of TGF-β signaling in this disease was demonstrated immunohistochemically by significantly higher nuclear SMAD2 phosphorylation observed in 20 MDS bone marrows when compared with 7 non MDS anemic controls (P < 0.0001, 2 Tailed T Test, Image Pro Plus software). This data along with high levels of membrane-bound and plasma TGF-β observed in MDS patients in previous studies support the development of therapeutics targeting the TGF-β signaling pathways in this disease. SD-208 is a novel, potent and specific inhibitor of TGF-β Receptor I (TGFβ-RI) kinase. We demonstrate that SD-208 blocks the phosphorylation of SMAD2 in hematopoietic progenitors which are at the colony forming unit-erythroid (CFU-E) stage of differentiation. SD-208 also abrogates the G0/G1 cell cycle arrest induced by TGF-β in bone marrow progenitors. SD-208 treatment leads to reversal of the myelosuppressive effects of TGF-β on erythroid and myeloid colony formation from primary human CD34+ cells. Selectivity of SD-208 in inhibiting TGF-β-mediated effects on hematopoiesis was supported by similar results observed with siRNAs targeting SMAD2, a major component of the TGF-b signaling pathway. Finally, the efficacy of SD-208 in MDS was evaluated by treating bone marrow mononuclear cells from 15 patients with early low grade MDS. SD-208 treatment led to dose-dependent increases in erythroid and myeloid colonies after 14 days of in vitro culture. The effect was most notable in patients with high levels of activated SMAD-2, as assessed by immunohistochemical staining of bone marrow biopsies. Stimulation of hematopoiesis in MDS-derived marrow culture by SD-208 demonstrates a novel concept and potential therapeutic role for TGFβ-RI inhibition in this disease. Supported by VISN-17 grant, Harris Methodist Foundation Grant and ASCO YIA to AV

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SGX70393 Inhibits Bcr-AblT315I In Vitro and In Vivo and Completely Suppresses Resistance When Combined with Nilotinib or Dasatinib.

Blood ◽

10.1182/blood.v110.11.535.535 ◽

2007 ◽

Vol 110 (11) ◽

pp. 535-535 ◽

Cited By ~ 3

Author(s):

Thomas O’Hare ◽

Christopher A. Eide ◽

Jeffrey W. Tyner ◽

Amie S. Corbin ◽

Matthew J. Wong ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Kinase Inhibitor ◽

Single Agent ◽

Resistance Profile ◽

Murine Bone Marrow ◽

Abl Kinase ◽

A Cell

Abstract Overview: Bcr-AblT315I is detected in the majority of CML patients who relapse after dasatinib- or nilotinib-based second-line Bcr-Abl kinase inhibitor therapy. SGX70393, an azapyridine-based Abl kinase inhibitor, is effective against Bcr-Abl and Bcr-AblT315I at low nanomolar concentrations in vitro and in cell lines. Here, we comprehensively profiled SGX70393 against native and mutant Bcr-Abl in vitro and in vivo. We also used a cell-based mutagenesis screen to evaluate the resistance profile of SGX70393 alone and in combination with imatinib, nilotinib, or dasatinib. Methods: We assessed colony formation in the presence of SGX70393 by murine bone marrow infected with retroviruses for expression of Bcr-Abl, Bcr-AblT315I, or a variety of other mutants. Toxicity was tested in clonogenic assays of normal bone marrow. SGX70393 effects on cellular tyrosine phosphorylation were measured by immunoblot and FACS in primary Bcr-AblT315I cells isolated from patients with CML or Ph+ B-ALL. In vivo activity was evaluated in a xenograft model using Ba/F3 cells expressing Bcr-AblT315I. Lastly, the resistance profile of SGX70393 was evaluated alone and in dual combinations with imatinib, nilotinib, or dasatinib in a cell-based mutagenesis assay. Results: Colony formation by murine bone marrow cells expressing Bcr-AblT315I (IC50: 180 nM) was reduced by SGX70393 in a dose dependent manner, while no toxicity was observed in colony forming assays of normal human or murine mononuclear cells at concentrations up to 2 μM. Ex vivo exposure of human Bcr-AblT315I mononuclear cells to SGX70393 decreased CrkL phosphorylation, while imatinib, nilotinib, or dasatinib had no effect. SGX70393 inhibited Bcr-AblT315I-driven tumor growth in mice and this was correlated with reduced levels of pCrkL in tumor tissue, while imatinib was ineffective. A cell-based mutagenesis screen revealed a profile of resistant clones confined to four p-loop residues and position 317. SGX70393 in combination with imatinib contracted the spectrum of resistant mutations relative to either single agent, though outgrowth could not be completely suppressed. Combining SGX70393 with low concentrations of nilotinib or dasatinib narrowed the resistance profile still further (residues 248 and 255 for nilotinib; 317 for dasatinib) and, with clinically achievable doses of either second drug, completely abrogated emergence of resistant subclones. Conclusions: SGX70393, a potent inhibitor of Bcr-AblT315I, exhibits a resistance profile centered around the p-loop and residue 317 of Bcr-Abl. Remarkably, in combination with nilotinib or dasatinib, outgrowth of resistant clones is completely suppressed. Single-agent therapy with an effective T315I inhibitor may provide a viable option for patients who relapse with Bcr-AblT315I. However, as a broader spectrum of mutations accounts for imatinib resistance, patients with acquired dasatinib or nilotinib resistance may continue to harbor residual mutant clones other than T315I. Thus, the full clinical potential of SGX70393 may be realized in combinations with a second Abl kinase inhibitor. Our findings provide the first demonstration that Abl kinase inhibitor combinations that include a T315I-targeted component such as SGX70393 have the potential to pre-empt Bcr-Abl-dependent resistance.

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Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽

10.1182/blood.v114.22.2531.2531 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2531-2531

Author(s):

Pani A. Apostolidis ◽

Stephan Lindsey ◽

William M. Miller ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Platelet Surface ◽

Platelet Counts ◽

Reticulated Platelets ◽

High Ploidy ◽

And Control ◽

Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

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Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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Morphological Distinction Unravels Mechanisms Of Platelet Biogenesis From Bone Marrow Megakaryocytes

Blood ◽

10.1182/blood.v122.21.2428.2428 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2428-2428

Author(s):

Satoshi Nishimura ◽

Koji Eto ◽

Ryozo Nagai

Keyword(s):

Bone Marrow ◽

Fluorescent Protein ◽

Conflicts Of Interest ◽

Human Subjects ◽

Blood Platelets ◽

Serum Levels ◽

Cytokine Balance ◽

Green Fluorescent

Abstract Blood platelets are generated in the bone marrow (BM) from their precursors, megakaryocytes (MK). Although we know that MKs produce platelets throughout life, precisely how platelets are produced in vivo remains uncertain, largely because of the rarity of MKs in the BM and the lack an adequate visualization technique. In the present study, we were able to visualize MK dynamics leading to platelet release in living animals at high resolution. To clearly understand the nature of thrombopoiesis in BM MKs, we optimized an in vivo imaging technique based on two-photon microscopy that enabled us to visualize living BM in CAG- enhanced green fluorescent protein (eGFP) mice. By visualizing living bone marrow in vivo, we observed that two modes (fragmentation and proplatelet formation) can be ongoing simultaneously in the same mouse. We observed that these two modes detectable by different morphological behavior can be ongoing simultaneously in the same BM of mouse, and are regulated by specific cytokines. Short proplatelets from megakaryocytes predominated at steady state, and more elongated proplatelets were accelerated by thrombopoietin (TPO) with responding to chronic platelet needs including recovery form BM transplantations. In contrast, acute platelet needs by blood loss, 5-FU administration or pritoneal acute inflammation increased cytoplasmic fragmentation following rapid ‘rupture’. Observed two modes are both dependent on tubulin reorganization on platelet biogenesis. In addition, platelet increase at acute phase is independent of proliferation by MK progenitors and this factor might exert apoptosis machinery on already reserved mature type of MKs. This humoral factor was identified by combination of in vitro screening systems and in vivo MK visualization analysis. Factor serum levels were reduced independently of the thrombopoietin level in human subjects with low platelet counts. It thus appears the cytokine balance dynamically regulates the mode of thrombopoiesis and the cellular programming of MKs. Thus, these novel factor may be a novel therapeutic target in thrombocytopenic situations, especially when associated with acute loss of platelets or when platelet transfusion is limited or unsuccessful. Disclosures: No relevant conflicts of interest to declare.

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Angptl-4 Is Upregulated Under Inflammatory Conditions In The BM Of Mice, Expands Myeloid Progenitors and Accelerates Reconstitution Of Platelets After Myelosuppressive Therapy

Blood ◽

10.1182/blood.v122.21.3677.3677 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3677-3677

Author(s):

Anne Schumacher ◽

Till Braunschweig ◽

Bernd Denecke ◽

Tim H. Brümmendorf ◽

Patrick Ziegler

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Relative Increase ◽

T Test ◽

Gm Csf ◽

Emergency Situations ◽

Inflammatory Conditions

Abstract The concerted action of hematopoiesis supporting cytokines such as G-CSF, GM-CSF or IL-6 regulates hematopoiesis during steady state and emergency situations. Respective knockout mice show defects both in production and function of myelopoietic effector cells. However, alternative pathways are likely to exist as mice with single or combined deficiencies for G-CSF, GM-CSF, and IL-6 or G-CSF and GM-CSF are still able to mount reactive neutrophilia responses during inflammatory conditions. In order to identify pathways for inflammation induced enhancement of hematopoiesis as well as to find new cytokines, which enhance myeloid cell regeneration, we analyzed the bone marrow (BM) of lipopolysaccharide (LPS) and vehicle injected wild type (WT) mice (single IP- injection) by gene expression microarray. Focusing on the identification of genes encoding for secreted or membrane proteins, we found 83 candidates to be up- and 14 to be downregulated after LPS treatment. Among known candiates, we found angiopoietin-like 4 (Angptl-4) as a predominantly upregulated gene in the BM of LPS-treated WT-mice. Upregulation was confirmed by RT-PCR as well as by Elisa in the BM of LPS treated mice and bone marrow stromal cells (BMSC) were identified as candidate producer cells. Functionally, we found recombinant Angptl-4 to stimulate the proliferation of myeloid colony-forming units (CFU) in vitro. In mice, repeated injections of Angptl-4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with Angptl-4 resulted in elevated platelet counts both in untreated animals and after myelosuppressive therapy. After lethal irradiation and transplantation of syngeneic BM cells repetitive injections of recombinant Angptl-4 for 5 consecutive days resulted in an accelerated reconstitution of platelets starting at day 8 after transplantation. The 50% pre-treatment platelet count was reached on day 14 in Angptl-4-treated animals as compared to day 21 for transplanted controls receiving no Angptl-4 (n=8; p=0.03, student´s T test). In contrast, transplantation of BM cells from Angptl-4 pre-treated donor mice had no effect on the recovery of platelets in this setting. The frequency of CD41lowCD61+ immature megakaryocytes was significantly increased in the BM of Angptl-4 injected as compared to control mice (27% vs 19% of total megakaryocytes; p= 0.008, student´s T test). Furthermore, bone marrow cytology revealed local accumulation of megakaryocytes carrying dysplastic features in Angptl-4 injected mice. In summary, our data suggest that Angptl-4 plays a complementary role on hematopoiesis during emergency situations like sepsis. The use of Angptl-4 in the setting of autologous stem cell transplantation could represent a potential approach to accelerate the reconstitution of megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.

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mTOR Kinase Inhibitors Enhance Efficacy of TKIs in Preclinical Models of Ph-like B-ALL

Blood ◽

10.1182/blood.v128.22.2763.2763 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2763-2763 ◽

Cited By ~ 1

Author(s):

Moran Gotesman ◽

Thanh-Trang T Vo ◽

Sharmila Mallya ◽

Qi Zhang ◽

Ce Shi ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Inhibitor Therapy ◽

Cellular Viability ◽

Murine Bone Marrow ◽

Pdx Models

Abstract Background and Rationale: B-lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. While event-free survival (EFS) exceeds 85% for most patients treated with contemporary therapy, outcomes are very poor for children who relapse, highlighting a need for new treatments. In particular, children with Philadelphia chromosome-like (Ph-like) B-ALL (who lack BCR-ABL1 rearrangement) have high rates of relapse and mortality with conventional chemotherapy. Transcriptional profiling and genomic sequencing of Ph-like ALL specimens have identified a variety of alterations that activate oncogenic kinase signaling, including rearrangements (R) of CRLF2, ABL1, and PDGFRB. Addition of the tyrosine kinase inhibitor (TKI) imatinib to chemotherapy has dramatically improved EFS for patients with BCR-ABL1-rearranged (Ph+) B-ALL, and it is hypothesized that TKI addition to therapy will similarly improve outcomes for patients with Ph-like ALL. Our prior preclinical studies in Ph+ B-ALL demonstrated enhanced efficacy of combining TKIs (imatinib or dasatinib) with mTOR kinase inhibitors (TOR-KIs) (Janes et al., Nature Medicine 2010; Janes et al, Leukemia2013). In the current studies, we hypothesized that dual kinase inhibitor therapy would have superior anti-leukemia cytotoxicity in Ph-like ALL and thus investigated combined TKI and TOR-KI treatment using patient-derived xenograft (PDX) models of childhood Ph-like ALL. Methods: For in vitro studies, viably cryopreserved leukemia cells from established ABL1-R Ph-like ALL PDX models (2 ETV6-ABL1) were incubated with the TKI dasatinib, TOR-KIs, or both TKI + TOR-KI for 72 hours prior to flow cytometric assessment of cellular viability via Annexin V and propidium iodide staining. Two chemically distinct TOR-KIs (MLN0128 or AZD2014) were used to confirm on-target effects. Additional primary ABL1-R or PDGFRB-R Ph-like ALL specimens were plated in methylcellulose without or with inhibitors in colony-forming assays. Phosphoflow cytometry (PFC) analysis of ALL cells incubated with inhibitors was also performed to measure the ability of TKIs and TOR-KIs to inhibit intracellular ABL1 and PI3K/mTOR signaling pathways. For in vivo studies, Ph-like ALL PDX models were treated with dasatinib, the TOR-KI AZD8055, or both drugs via daily oral gavage for 8 days. Human CD19+ ALL was quantified in murine spleens and bone marrow at end of treatment with quantification of cycling cells by EdU incorporation. PFC analysis of murine bone marrow was also performed 2 hours after drugs were dosed, to measure in vivo inhibition of signaling proteins. Results: Combined in vitro treatment with dasatinib and MLN0128 or AZD2014 decreased cellular viability more than inhibitor monotherapy. Similarly, in a set of CRLF2-rearranged samples, mTOR inhibitors augmented killing by the JAK2 inhibitor BBT-594. Incubation of primary ABL1-R or PDGFRB-R ALL cells with both dasatinib and AZD2014 more robustly inhibited colony formation than did inhibitor monotherapy. In in vitro PFC analyses of ABL1-R samples, we observed expected dasatinib-induced inhibition of phosphorylated (p) STAT5. Inhibition of the mTOR substrate pS6 was observed with dasatinib, MLN0128, and AZD2014 with more complete inhibition achieved when dasatinib combined with either MLN0128 or AZD2014. Similarly, in vivo treatment of PDX models with dasatinib and AZD8055 reduced leukemia burden and pS6 signaling more completely than either inhibitor alone. Importantly, dual inhibition decreased the percentage of cycling human ALL cells in murine bone marrow, but preserved cycling in normal mouse bone marrow cells in the same animals. Our data thus provide additional compelling preclinical rationale for combined inhibitor therapy with TKIs and TOR-KIs in Ph-like ALL. Disclosures Weinstock: Novartis: Consultancy, Research Funding. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

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Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽

10.1182/blood-2019-122449 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2488-2488 ◽

Cited By ~ 1

Author(s):

José Gabriel Barcia Durán

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Fluorescent Microscopy ◽

Hematopoietic Stem ◽

Interleukin Receptors ◽

Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

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Expansion of senescent megakaryocyte-lineage cells maintains CML cell leukemogenesis

Blood Advances ◽

10.1182/bloodadvances.2020003117 ◽

2020 ◽

Vol 4 (24) ◽

pp. 6175-6188

Author(s):

Yamato Tanabe ◽

Shimpei Kawamoto ◽

Tomoiku Takaku ◽

Soji Morishita ◽

Atsushi Hirao ◽

...

Keyword(s):

Bone Marrow ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

Fusion Gene ◽

Cell Number ◽

Hematopoietic Stem ◽

Enhanced Production ◽

Tgf Β1

Abstract BCR-ABL, an oncogenic fusion gene, plays a central role in the pathogenesis of chronic myeloid leukemia (CML). Oncogenic signaling induces oncogene-induced senescence and senescence-associated secretory phenotype (SASP), which is characterized by enhanced production of various cytokines. BCR-ABL gene transduction confers senescent phenotype in vitro; however, the in vivo relevance of senescence has not been explored in this context. Transplantation of BCR-ABL–expressing hematopoietic stem/progenitor cells caused CML in mice with an increase in bone marrow BCR-ABL+CD41+CD150+ leukemic megakaryocyte-lineage (MgkL) cells, which exhibited enhanced senescence-associated β-galactosidase staining and increased expression of p16 and p21, key molecules that are crucially involved in senescence. Moreover, knockout of p16 and p21 genes reduced both BCR-ABL–induced abnormal megakaryopoiesis and the maintenance of CML cell leukemogenic capacity, as evidenced by attenuated leukemogenic capacity at secondary transplantation. The expression of transforming growth factor-β1 (TGF-β1), a representative SASP molecule, was enhanced in the leukemic MgkL cells, and TGF-β1 inhibition attenuated CML cell leukemogenic capacity both in vitro and in vivo. Furthermore, BCR-ABL–expressing MgkL cells displayed enhanced autophagic activity, and autophagy inhibition reduced bone marrow MgkL cell number and prolonged the survival of CML mice, which had transiently received the tyrosine kinase inhibitor, imatinib, earlier. Thus, BCR-ABL induced the expansion of senescent leukemic MgkL cells, which supported CML leukemogenesis by providing TGF-β1.

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