scholarly journals Antileukemic Activity of 8-Chloro-Adenosine (8-Cl-Ado) Is Mediated By Mir-155 Degradation and ErbB3 Binding Protein (Ebp1)-Dependent p53 Activation: A Novel Therapeutic Approach for FLT3-ITD Acute Myeloid Leukemia (AML)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3938-3938
Author(s):  
Ralf Buettner ◽  
Le Xuan Truong Nguyen ◽  
Bijender Kumar ◽  
Corey James Morales ◽  
Chao Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Growth ◽  
Research Funding ◽  
Leukemia Cell ◽  
Antileukemic Activity ◽  
Down Regulation ◽  
P53 Expression ◽  
Blast Cells ◽  
P53 Activation ◽  
Novel Therapeutic

Abstract AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values <1 at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90)]. In an orthotopic mouse model where FLT3-ITD-positive MV4-11 cells were xenografted, mice treated with 75 mg 8-Cl-Ado/kg/day plus quizartinib (0.5 mg/kg/day) survived significantly longer than mice treated with the individual agents or vehicle alone (median survival 54.2 days vs. 27.3 days for controls, p=0.016). Taken together, these results support 8-Cl-Ado as a promising novel agent via a unique mechanism of action for FLT3-ITD AML, mediated by miR-155 degradation. 8-Cl-Ado also synergizes with TKIs both in vitro and in vivo thereby representing a potentially novel clinical approach for FLT3-ITD AML. A single-agent phase I/II clinical trial in relapsed/refractory AML is already ongoing at our institution. Figure. Figure. Disclosures Wennerberg: Novartis: Research Funding. Gandhi:Pharmacyclics: Research Funding.

scholarly journals RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2187-2187
Author(s):  
Xiaomei Yan ◽  
Yoshihiro Hayashi ◽  
Xinghui Zhao ◽  
Aili Chen ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Down Regulation ◽  
Leukemia Cell Lines ◽  
Flanking Regions ◽  
The Impact

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

Essential Role for PU.1 in MEIS1 Activation and MLL Fusion Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3466-3466
Author(s):  
Jing Zhou ◽  
Bo Li ◽  
Jun Wu ◽  
Fuhong He ◽  
Qiang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Essential Role ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Genetic Alterations ◽  
Regulation Of Transcription ◽  
Mouse Bone Marrow ◽  
Down Regulation ◽  
Hematopoietic Stem

Abstract Abstract 3466 Down-regulation of transcription factor PU.1, a key regulator of hematopoiesis, induces myeloid leukemia in mice, demonstrating a role of PU.1 as tumor suppressor. Recent studies, however, have also suggested that PU.1 is required for repopulation/self-renewal capacity of normal hematopoietic stem cells (HSCs), and presence of PU.1 activity may be necessary to favor growth of myeloid leukemia stem cells. To explore whether PU.1 could possibly act as an oncogene in the development of certain type of myeloid leukemia, we set to look for differential up-regulation of PU.1 among AML patients with distinct cytogenetic and genetic alterations in public databases. Consistent with recent molecular studies showing suppression of PU.1 expression by AML1-ETO and PML-RARa fusion proteins, PU.1 expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. In contrast, PU.1 expression level in MLL leukemia patients is significantly higher than that of other subgroups of AML. In addition, we found that a set of PU.1 direct target genes, as defined by genome wide location analysis of this factor, expresses at higher level in MLL leukemia patients comparing with those with t(8;21) and t(15;17) translocations, supporting an increased PU.1 activity in this subgroup of leukemia. In our effort to characterize the functional consequence of high expression of PU.1 in AML, we found that PU.1 plays an essential role in activation of MEIS1, an oncogene essential for MLL leukemia stem cell potential, and in development of MLL fusion leukemia. MEIS1, as PU.1, is differentially up-regulated in MLL leukemia patients, and expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. Among AML patients with higher level MEIS1 expression, a positive correlation was observed between expression of PU.1 and that of MEIS1. Using promoter reporter assay, electro mobility shift assay (EMSA) and chromatin immunoprecipiation (ChIP) analysis, we found that PU.1 directly binds to and activates MEIS1 promoter in vitro and in vivo. Analysis of a hypomorphic PU.1 mouse model indicated that PU.1 is required to maintain Meis1 expression in murine HSCs and progenitors, and knockdown of PU.1 in patient-derived MLL leukemia cell lines resulted in lower enrichment of PU.1 protein at MEIS1 promoter, accompanied by down-regulation of MEIS1 expression and decreased proliferation and survival of these cells. We are now examining whether the ability of MLL-AF9 fusion protein to drive leukemia is compromised in PU.1-deficient mouse HSC/HPCs, and whether introduction of exogenous Meis1 can compensate for the loss of PU.1 in the development of MLL-AF9 leukemia in mouse bone marrow transplantation model. Finally, we are also testing knock-down of PU.1 as a therapeutic approach to primary AMLs isolated from MLL leukemia patients. Collectively, our data indicate that PU.1 is required for the pathogenesis of MLL associated leukemia, at least partially, through direct activation of MEIS1. In veiw of the dependency of MEIS1 in MLL leukemic transformation, targeting PU.1 mediated MEIS1 gene activation could be an alternative or synergistic approach for MLL leukemia therapies aimed at inhibition of DOT1L and HOXA9. Disclosures: No relevant conflicts of interest to declare.

PF-04449913 Reverts Multi Drug Resistance (MDR) by a Strong Down-Regulation of ABCA2 and BCL2 on Leukemia Stem Cells in Phase I Acute Myeloid Leukemia and Chronic Myeloid Leukemia Treated Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1429-1429 ◽  
Author(s):  
Cristina Papayannidis ◽  
Viviana Guadagnuolo ◽  
Ilaria Iacobucci ◽  
Sandra Durante ◽  
Carolina Terragna ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Fold Change ◽  
P Value ◽  
Atp Binding ◽  
Down Regulation ◽  
Before And After ◽  
Equity Ownership ◽  
Atp Binding Cassette

Abstract Abstract 1429 The development of resistance against chemotherapy remains one of the major challenges in the clinical management of leukemia. The Sonic Hedgehog (Hh) pathway is an essential regulator of multidrug resistance (MDR) in leukemia by exerting it's effect on leukaemia stem cells (LSC). PF-04449913 is a selective and potent small molecule inhibitor of the Hh pathway and leukemia self-renewal and is currently being evaluated in Phase I clinical trials. In order to evaluate the activity of PF-0444913 in overcoming the drug resistance of leukemia stem cells, we studied leukemia stem cell population (CD34+ subpopulation) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies including Myelofibrosis (MF), MDS, Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic leukemia (CMML) and Acute Myeloid Leukemia (AML) patients. We were able to collect and separate highly purified (98%) bone marrow hematopoietic progenitor cells (CD34+ populations) in 5 AML, 1 MF and 2 CML patients, by immunomagnetic separation, and analyze them for gene expression profile (GEP) using Affimetrix HG-U133 Plus 2.0 platform. We have observed that 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Among these genes, we demonstrated a down regulation of Bcl2 (fold change −1.03004; p value= 0.01), ABCA2 (fold change −1.08966; p value=0.03), Bcl2l13 (fold change −1,04259; p-value=0,027642), Bcl2l2 (fold change −1,17214; p-value=0,000768), Casp4 (fold change −1,06551; p-value=0,032428), Casp7 (fold change −1,01569; p-value=0,006688), Casp10 (fold-change −1,3076; p-value=0,050431), ABCF1 (fold change −1,04999; p-value=0,07213). On the contrary, ABCB1 (fold change 1,46592) and ABCG2 (fold change −1,16103) are respectively up and down regulated, with a not statistically significant p-value (0,35375 and 0,288194 respectively). Bcl2 (B-cell lymphoma 2), Bcl2l2 (Bcl2-like protein 2) and Bcl2l13 (Bcl2-like 13) are the founding members of the Bcl-2 family of apoptosis regulator proteins. Recent studies showed that Hh signals upregulate Bcl2 to promote cellular survival. Casp 4,7,10 (Caspases, or c ysteine-asp artic proteases) are a family of cysteine proteases that play essential roles in apoptosis, necrosis, and inflammation. ABCA2 (ATP-binding cassette sub-family A member 2), ABCF1 (ATP-binding cassette sub-family F member 1), ABCB1 (ATP-binding cassette sub-family B member 1, MDR1), ABCG2 (ATP-binding cassette sub-family G member 2) belong to the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intracellular membranes. One mechanism of MDR is the increased expression of ABC drug transporters that mediate energy-dependent transport of drugs out of the cells against a concentration gradient, resulting in low intracellular drug concentrations. This is a common finding in LSC, and represents an important clinical problem for disease eradication. Furthermore, we evaluated Gli1, Gli2 and Smo expression by GEP, comparing data before and after 28 days of treatment with PF-04449913 and, as expected, we observed a down regulation of Gli1 (fold change −1.0775), Smo (fold change −1.07702), and an up regulation of Gli2 (fold change 1.08191). Our results suggest that PF-04449913 is able to revert MRD mechanisms of LSC by a strong down regulation of genes (Bcl-2, Bcl-2l13, Bcl-2l2, ABCA2, and ABCF1), which are critical for chemoresistance in acute and chronic leukemia patients. Therefore, the combination of PF-04449913 with Tyrosine Kinase inhibitors or conventional chemotherapy could represent a valid new therapeutic approach in these haematological malignancies. Acknowledgments. Work supported by Pfizer, European LeukemiaNet, FIRB 2008, PRIN 2009, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Disclosures: Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Courtney:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Baccarani:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jamieson:Wintherix: Equity Ownership; Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria. Cortes:Novartis: Consultancy; Novartis: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. McLachlan:Pfizer: Employment. Van Arsdale:Pfizer: Employment. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria.

scholarly journals Immunoglobulin Superfamily Member 8 Is Indispensable for Myeloid Leukemia Via Wnt/β-Catenin Signaling Pathway

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 23-24
Author(s):  
Koji Jimbo ◽  
Takaaki Konuma ◽  
Takahiro Ito ◽  
Yaeko Nakajima-Takagi ◽  
Atsushi Iwama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Immunoglobulin Superfamily ◽  
Hematopoietic Stem

Immunoglobulin superfamily member 8 (IGSF8, also known as EWI-2, PGRL, and CD316), is a cell surface protein containing 4 immunoglobulin domains. IGSF8 directly binds to the tetraspanin molecules, CD9 and CD81, and modulates cell adhesion, migration, and growth. Previous studies demonstrated that IGSF8 was associated with prognosis and metastasis in several solid tumors. However, the role of IGSF8 in normal hematopoiesis and myeloid leukemia is still unclear. First, we examined the expression levels of Igsf8 in various hematopoietic fraction of wild-type murine bone marrow cells, and found that Igsf8 is expressed in all hematopoietic lineages. To investigate hematopoietic functions of Igsf8, we generated hematopoietic cells specific Igsf8 deleted mice (Igsf8fl/fl; Vav-Cre) and tamoxifen induced Igsf8 deleted mice (Igsf8fl/fl; Rosa26-CreERT). Igsf8fl/fl, Vav-Cre (denoted as Igsf8-/-) mice represented normal maturation. Deletion of Igsf8 did not significantly affect adult hematopoiesis in peripheral blood and bone marrow. Igsf8-/- long-term hematopoietic stem cells (LT-HSCs: CD34- Flk2- c-Kit+ Sca-1+ Lineage- cells) reduced colony forming ability in vitro, and serial competitive transplantation assay showed comparable donor chimerism by 3 months, but led to decrease Igsf8-/- donor chimerism at 4 months and those after second transplantation in vivo. These results suggest that Igsf8 does not affect the adult hematopoiesis, but it can affect their proliferative and reconstitutive capacity of HSCs. To investigate the effects of Igsf8 on myeloid leukemia, we generated MLL-AF9 and NRASG12V-driven acute myelogenous leukemia (AML), or BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) mice models. Igsf8-/- led to a dramatic reduction in the number of leukemic colonies formed in vitro (Figure 1A). Igsf8-/- leukemia mice showed significantly longer survival in vivo (Figure 1B). This effect was also observed by eliminating Igsf8 expression after leukemia establishment using conditionally deletion. Igsf8-/- AML cells showed decreased S phase fraction. Igsf8-/- leukemia stem cells (LSCs: c-Kit+ Lineage- cells) triggered an increment of the apoptosis, which contribute to significantly lower proportion of LSCs in spleen of Igsf8-/- leukemic mice. Given that Igsf8-/- did not affect homing ability of leukemia cells, these results indicate that Igsf8 is required for propagation of myeloid leukemia and maintenance of LSC. To understand the Igsf8-mediated regulation of myeloid leukemia, we conducted RNA sequencing analysis of LT-HSCs, and LSCs of AML and CML-BC. Gene set enrichment analysis exhibited increase apoptosis related genes and decrease Wnt/β-catenin related genes in Igsf8-/- leukemic cells, but not in LT-HSCs (Figure 1C). Increment of pro-apoptosis genes, and decrement of anti-apoptosis genes and Wnt/β-catenin target genes in Igsf8-/- AML stem cells were validated in quantitative polymerase chain reaction analysis. Further, expression levels of β-catenin protein in Igsf8-/- leukemic cells were significantly lower compared to Igsf8+/+ leukemic cells, but not in normal hematopoietic stem and progenitor cells (Figure 1D). These results suggest that Igsf8 might be critical for myeloid leukemia maintenance via Wnt/β-catenin signaling pathway. Then, we investigated the effects of IGSF8 on human myeloid leukemia. We confirmed IGSF8 expression in several human myeloid leukemia cell line and primary patient-derived leukemia cells. Knockdown of IGSF8 by small hairpin RNA in myeloid leukemia cell lines (THP-1, MV4-11, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro. Knockdown of IGSF8 also caused decrease expression of β-CATENIN in AML cell lines. These results indicate that IGSF8 is also required for propagation of human myeloid leukemia cells. Taken together, our present study reveals that Igsf8 is indispensable for myeloid leukemia, but not adult hematopoiesis, suggesting that IGSF8 inhibition should be considered for targeting myeloid leukemia. Disclosures Jimbo: Japan Society for the Promotion of Science: Research Funding. Konuma:SGH Foundation: Research Funding; The Japanese Society of Hematology: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Ito:Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

scholarly journals Role of Enhanced Autophagy in Resistance of FLT3-ITD AML Stem Cells to FLT3 TKI Treatment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1358-1358
Author(s):  
Shaowei Qiu ◽  
Andrew J Paterson ◽  
Ajay Abraham ◽  
Jianbo He ◽  
Mansi Shah ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Growth ◽  
Cellular Response ◽  
Conflicts Of Interest ◽  
Autophagic Flux ◽  
Independent Manner ◽  
P53 Expression ◽  
Tki Resistance ◽  
Tki Treatment ◽  
Autophagy Inhibition

Abstract The FLT3-ITD mutation occurs in 25-30% of AML patients, and is associated with increased relapse rate and poor survival. FLT3 tyrosine kinase inhibitors (TKI) have shown encouraging results, but responses are not sustained in most patients. Therefore there is considerable interest in understanding mechanism contributing to resistance and preservation of FLT3-ITD AML stem cells after TKI treatment. Autophagy is an important cellular response that maintains cell survival under stress. Here we investigated the regulation of autophagy in FLT3-ITD+ AML cells, and its role in resistance to FLT3 TKI treatment. Expression of a FLT3-ITD construct in FLT3-ITD negative OCI-AML3 cells using lentiviral vectors resulted in increased autophagic flux compared with wild-type FLT3 or non-transduced controls. However, treatment of FLT3-ITD+ MV4-11 and Molm13 AML cells, or primary FLT3-ITD+ CD34+ cells, with the potent FLT3 TKI AC220 (Quizartinib) did not significantly alter autophagic flux. These results suggest that FLT3-ITD expression increases autophagy but in a kinase-independent manner. MV4-11 and Molm13 cells were transduced with the GFP-LC3-RFP-LC3 ΔG construct (Kaizuka T, et al,2016) to measure autophagic flux. Autophagyhigh AML cells showed lower growth rate compared with Autophagylow cells (0.65, p=0.025), and reduced sensitivity to inhibition by AC220 (0.49 vs 0.71, p=0.003). Treatment of MV4-11 and Molm13 cells with the potent autophagy inhibitor Lys05 in combination with AC220 resulted in significantly enhanced inhibition of AML cell growth (MV4-11: 0.83 vs 0.54, p<0.001; Molm13: 0.58 vs 0.43, p=0.02) and increased induction of apoptosis (MV4-11: 0.81 vs 0.54, p<0.001; Molm13: 0.73 vs 0.49, p=0.024) compared to AC220 alone. We further show that inhibition of autophagy by shRNA-mediated knockdown of the critical autophagy gene ATG5 enhanced sensitivity of AML cells to AC220 treatment (MV4-11: 0.91 vs 0.65, p=0.005; Molm13: 0.88 vs 0.64, p=0.004). Treatment with Lys05 also inhibited autophagic flux in primary FLT3-ITD+ AML CD34+ stem/progenitor cells and resulted in significantly increased inhibition of cell cycle arrest (G0 stage: 40.3% vs 61.8%), increased myeloid differentiation (CD11b: 1 vs 1.24, p=0.009; CD14: 1 vs 1.21, p=0.0024), and increased cell death when combined with AC220 (0.58% vs 0.78%, p=0.0245). These results indicate that increased autophagy in FLT3-ITD+ AML cells is associated with TKI resistance, and that autophagy inhibition enhances sensitivity of FLT3-ITD+ AML cells to TKI treatment. Induction of autophagy by starvation or mTOR inhibitor treatment in MOLM13 and MV4-11 cells was associated with reduced p53 expression, whereas autophagy inhibition by chloroquine or SAR405, a PIK3C3/Vps34 inhibitor, was associated with increased p53 expression. Starvation-mediated autophagy in FLT3-ITD+ AML cells was significantly increased upon p53 knockdown, suggesting that p53 has an inhibitory effect on autophagy. Enhanced inhibition of FLT3-ITD+ AML cell growth by autophagy inhibition in combination with AC220 treatment was prevented by shRNA-mediated knockdown of p53, suggesting that effects of autophagy inhibition on FLT3-ITD+ AML cell growth were p53 dependent. We conclude that FLT3-ITD expression enhances autophagic flux in FLT3-ITD+ AML cells in a kinase-independent manner, and that increased autophagy contributes to TKI resistance of FLT3-ITD+ AML cells. We show that autophagy inhibition can enhance sensitivity of FLT3-ITD+ AML stem cells to TKI treatment. Finally, we demonstrate important roles for autophagy in regulating p53 levels, and conversely for p53 in regulating autophagy and the effects of autophagy inhibition in FLT3-ITD+ AML cells. These studies support further development of strategies for targeting of autophagy and related pathways to enhance efficacy of TKI treatment in eliminating AML stem cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of Autophagy in Resistance of FLT3-ITD AML Stem Cells to FLT3 TKI Treatment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2548-2548
Author(s):  
Shaowei Qiu ◽  
Chengcheng Yan ◽  
Andrew J Paterson ◽  
Hui Li ◽  
Nick Anderson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Growth ◽  
Autophagic Flux ◽  
P53 Expression ◽  
Enhanced Sensitivity ◽  
Cd34 Cells ◽  
Tki Treatment ◽  
Autophagy Inhibition

The FLT3-ITD mutation occurs in 25-30% of AML patients, and is associated with increased relapse rate and poor survival. FLT3 tyrosine kinase inhibitors (TKI) have shown encouraging results, but responses are not sustained. Therefore, there is considerable interest in understanding mechanisms of resistance of FLT3-ITD AML stem cells after TKI treatment. Autophagy is an important cellular response that maintains cell survival under stress. Here we investigated the regulation of autophagy in resistance of FLT3-ITD+ AML stem cells to FLT3 TKI treatment. MV4-11 and Molm13 FLT3-ITD AML cells were transduced with GFP-LC3-RFP-LC3 ΔG constructs to measure autophagic flux. Autophagyhigh AML cells showed slower growth(p<0.0001), enhanced sensitivity to the autophagy inhibitor Lys05 (p=0.01) and reduced sensitivity to the FLT3-ITD inhibitor AC220 (p=0.03) compared with autophagylow cells. Treatment with Lys05 in combination with AC220 resulted in significantly enhanced inhibition of AML cell growth (p=0.02) and increased induction of apoptosis (p=0.024) compared to AC220 alone. We confirmed that inhibition of autophagy by shRNA-mediated knockdown of the critical autophagy gene ATG5 also enhanced sensitivity of AML cells to AC220 treatment (p=0.004). Lys05 treatment also inhibited autophagic flux in primary human FLT3-ITD+ AML CD34+ stem/progenitor cells, and resulted in significantly increased apoptosis (78% vs 58%, p=0.0245) and reduced cell growth (47% vs 68%, p=0.003) when combined with AC220 compared to AC220 alone. In contrast Lys05 treatment had minimal effect on normal CD34+ stem cells, indicating that autophagy inhibition selectively enhances sensitivity of FLT3-ITD+ AML CD34+ cells to TKI treatment while sparing normal CD34+ cells. We tested the effect of Lys05 in vivo by treating NSG mice engrafted with primary FLT3-ITD AML cells with vehicle, AC220 (10mg/kg), Lys05 (20mg/kg) or combination for 3 weeks. Lys05 treatment reduced human CD45+ cell engraftment (8.7% vs 17%, p=0.03), and reduced CD45+CD33+ myeloid cells in BM and spleen (BM: 42% vs 81%, p<0.001, Spleen: 8% vs 14%, p=0.01), compared with vehicle. The combination of AC220 with Lys05 reduced CD45 engraftment (11% vs 37%, p=0.004) and absolute CD45+CD34+ stem cell numbers in BM compared with AC220 alone. The effect of Lys05 in vivo was also investigated in a transgenic FLT3-ITD AML mouse model (Mx1-cre-Tet2f/f, Flt3-ITDKi). The ability of in vivo Lys05 treatment to inhibit autophagy was confirmed by immunofluorescence. Lys05 inhibited autophagy flux to a greater extent in c-Kit+ stem/progenitor cells compared to differentiated cells. Leukemic mice were treated with PBS, Lys05(20mg/kg), AC220(10mg/kg) or the combination for 3 weeks. The combination of AC220 and Lys05 resulted in significantly greater reduction of WBC and neutrophil counts compared to the other groups(p=0.03). The AC220 and Lys05 combination reduced spleen weight and cellularity significantly more than AC220 alone (p=0.02), and splenic ST-HSC, MPP, GMP, MEP and BM GMP, MEP significantly more than other groups. However, BM ST-HSC and MPP (LT-HSC are absent in this AML model) were similar among the different treatment groups. The proportion of mature myeloid and T cells was increased in the combination group. Ki67 and Dapi labeling indicated that Lys05 reduced the frequency of ST-HSC and MPP in G0 and increased those in G1. The ability of Lys05 to promote quiescent LSC entry into cell cycle may contribute to enhanced sensitivity to AC220 when the two are used in combination. We show that induction of autophagy in MOLM13 cells by starvation and mTOR inhibitor was associated with reduced p53 expression, whereas autophagy inhibition by chloroquine or SAR405, a PIK3C3/Vps34 inhibitor, was associated with increased p53 expression by WB. Inhibition of growth or increased apoptosis in FLT3-ITD+ AML cells following autophagy inhibition were prevented by shRNA-mediated knockdown of p53, suggesting that effects of autophagy inhibition on FLT3-ITD+ AML cell growth were p53 dependent. We conclude that autophagy inhibition enhances sensitivity of FLT3-ITD+ AML stem cells to TKI treatment both in vitro and in vivo. We demonstrate important roles for autophagy in regulating p53 levels and cell cycle in FL3-ITD AML cells. These studies support further development of strategies to target autophagy and related pathways to enhance efficacy of TKI in eliminating AML stem cells. Disclosures No relevant conflicts of interest to declare.

The Expansion Of CML Clones Initiates At The CMP Stage, and Is Associated With The Down-Regulation Of IRF8 and GFI1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1477-1477 ◽  
Author(s):  
Kohta Miyawaki ◽  
Hirotake Kusumoto ◽  
Tadafumi Iino ◽  
Kentaro Kohno ◽  
Hirofumi Tsuzuki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Tyrosine Kinase ◽  
Leukemic Stem Cells ◽  
Positive Clone ◽  
Down Regulation ◽  
Dependent Cell ◽  
Myeloid Progenitors

Abstract In the chronic phase of chronic myeloid leukemia (CML-CP), leukemic stem cells do not necessarily depend on the BCR-ABL tyrosine kinase activity for their growth and survival and thus resistant to tyrosine kinase inhibitors (TKIs). In this study, we aimed to identify the initial progenitor population that is getting switched on BCR-ABL growth signaling and tried to elucidate the underlying molecular mechanisms of BCR-ABL dependent cell growth. We thus intensively analyzed the involvement status of CML clones in each developmental stage at diagnosis. To identify the hematopoietic stem or progenitor cell stage that is responsible for CML clone expansion, bone marrow cells from 13 newly-diagnosed CML-CP patients were analyzed by FACS, and purified stem/progenitor populations were tested for the t(9;22) involvement by FISH. Gene expression signature of each purified population was also evaluated by cDNA microarray. CD34+CD38- HSC fraction was markedly diminished in all CML-CP patients compared to healthy volunteers (<1% of CD34+ cells in patients vs. ∼10% in volunteers), whereas CD34+CD38+ myeloid progenitors expanded. Interestingly, the t(9;22) positive ratios in the HSC fraction were greatly diversified among patients (Figure 1). Of note, in 4 patients, the involvement of t(9;22) positive clone was less than 10%, suggesting that their leukemic stem cells have not outgrown normal HSCs. Among CD34+CD38+ myeloid progenitors, common myeloid progenitors (CMPs) robustly expanded and were composed more than 90% of t(9;22) positive clone in all cases. Downstream of CMPs, megakaryocyte/erythrocyte progenitors (MEPs) but not granulocyte/macrophage progenitors (GMPs) were dominantly involved in leukemia (t(9;22) positive ratio; 96.0+/-5.5% vs. 56.3+/-37%). The expression level of BCR-ABL is not different among these progenitor populations. These observations collectively suggest that in CMP-CP, BCR-ABL signaling becomes effective on cell proliferation especially at the CMP stage. Gene expression analysis of stem/progenitor populations in CML patients revealed that IRF8 and GFI1, transcription factors playing critical roles in myeloid differentiation and cell proliferation, were down-regulated specifically in CMPs as compared with that in normal controls (Figure 2). In order to substantiate the role of IRF8 and GFI1 in CML pathogenesis, we used a CML mouse model established by enforced retroviral expression of BCR-ABL. As in analysis of CML patients, BCR-ABL expressing CMPs but not stem/multipotent progenitor cells acquired growth advantage over normal counterparts. Importantly, the expression of IRF8 and GFI1 became undetectable after BCR-ABL transduction in the expanding CMPs. Our observations revealed that, in CML-CP hematopoiesis, BCR-ABL dependent cell proliferation initiates at the CMP stage, and is accompanied with the down–regulation of IRF8 and GFI1. Because IRF8 knockout mice develop myeloproliferative disorders, and because CMPs expand in GFI1 null mice, the attenuation of these molecules could be downstream effector of BCR-ABL dependent myeloid cell growth. Taken together, the reactivation of these molecules might be useful to develop alternative therapeutic strategies for CML-CP, for example, with TKI-resistant BCR-ABL mutants. Disclosures: Miyamoto: Kyushu University Hospital: Employment.

Chlorpromazine, an Inhibitor of Intracellular Trafficking of FLT3-ITD and KIT D816V, Shows Prominent Anti-Leukemic Activities Against AML Cells and AML Stem Cells in Vitro and in Vivo.

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 269-269 ◽  
Author(s):  
Shinya Rai ◽  
Hirokazu Tanaka ◽  
Mai Suzuki ◽  
Akira Tanimura ◽  
Keiko Matsui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Normal Saline ◽  
Intracellular Trafficking ◽  
Research Funding ◽  
Leukemia Cell ◽  
Growth And Survival ◽  
Leukemia Cell Lines ◽  
Kit D816v

Abstract Activating mutations of receptor tyrosine kinases (RTKs) are involved in the pathophysiology of various types of cancers. Among them, FLT3 internal tandem duplications (ITD) and a KIT point mutation at D816V are frequently found in acute myeloid leukemia (AML) and associated with poor prognosis. Although a number of clinical trials were conducted, an effective RTK inhibitor with acceptable toxicity hasn’t been developed. Recent studies have demonstrated these oncogenic RTKs are mislocalized in the cytoplasm, where they transmit aberrant leukemogenic signals to downstream molecules in a compartment-specific manner. So, we speculated that manipulation of their intracellular trafficking between intracellular compartments might be promising as a new therapeutic approach for AML with mutant RTKs. In this study, we evaluated the in vitro and in vivo anti-leukemic effects of chlorpromazine (CPZ) as an inhibitor of membrane trafficking, which is a well-established antipsychotic drug. All of the protocols were approved by the Institutional Review Board of Kinki University Faculty of Medicine, and we obtained AML samples after the written informed consent was given from all the patients. First, to examine the effect of CPZ on the intracellular trafficking of RTK mutants, we introduced murine FLT3-ITD and KIT-D814V (corresponding to human KIT D816V) into murine embryonic fibroblasts (MEFs) isolated from E14.5 fetuses, respectively. Both FLT3-ITD and KIT-D814V were distributed in cytoplasm with a punctate pattern, both of which were mainly located close to endoplasmic reticulum (ER) and Golgi. In contrast, when treated with CPZ, both RTK mutants were predominantly localized in the compartment apart from ER and Golgi in a diffused pattern, indicating that the intracellular trafficking system that retains mutated RTKs at ER or Golgi is disrupted by CPZ treatment. Next, we evaluated the effect of CPZ on cell growth and survival using several human leukemia cell lines and primary AML cells with or without mutant RTK. As a result, CPZ severely inhibited the growth and survival of leukemia cell lines and primary AML cells with mutant RTK in a dose-dependent manner (up to 95% inhibition; IC50: between 1.5 μM and 10.7 μM). In addition, CPZ inhibited the growth of the cells without mutant RTK, whereas the IC50 levels were 3- to 4-fold higher than those seen in the cells with mutant RTK. We also examined the effects of CPZ on the growth and survival of CD34+38- leukemic stem cells (LSCs) derived from AML patients (n=4), and found that CPZ effectively inhibited the growth and/or survival of LSCs, especially in LSCs with mutant RTK (IC50=7.5 μM). As for this mechanism, we found that CPZ significantly suppressed the activities of mutant RTKs and their downstream molecules such as Stat5 in all tested FLT3-ITD(+) LSCs. These results indicate that CPZ inhibits cell growth and/or survival of AML cells and LSCs harboring mutant RTKs. Next, we evaluated the in vivo anti-leukemic effect of CPZ with a xenograft model. Three days after the transplantation of human AML cells with mutant RTKs (n=8), NOD/Scid/IL2Rg-KO (NOG) mice were treated with normal saline or 10 mg/kg CPZ intraperitoneally on everyday schedule. At first, we confirmed that plasma concentration levels of CPZ on this schedule were within the non-toxic range as previously reported (0.03 to 0.71 μM, n=3) and that CPZ doesn’t affect normal hematopoiesis (n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ AML cells and CD34+38- LSCs were significantly reduced by CPZ treatment in the bone marrow (BM) of the recipient mice compared with normal saline-treated control (ctrl) mice (CD45+ 58.2% vs 8.47%, p<.001; LSCs 0.51% vs 0.07%, p<.005), indicating that CPZ can inhibit the growth and survival of both AML cells and LSCs in vivo. We further tested the anti-leukemic activities of CPZ on established human AML in a zenograft model. After confirming the engraftment of human AML cells by BM sampling six weeks after transplantation of FLT3-ITD(+) AML cells (n=3), these mice were treated with CPZ or normal saline. In all cases tested, CPZ treatment for 4 weeks significantly reduced human AML fraction in the BM. (18.4% vs 1.99%, p<.005) Together, these results suggest that CPZ, an inhibitor of the intracellular trafficking of leukemogenic RTKs, would be a promising therapeutic drug to eradicate AML cells with an established safety profile. Disclosures Rai: Shionogi & Co., Ltd. : Research Funding. Tanaka:Shionogi & Co., Ltd. : Research Funding. Tanimura:Shionogi & Co., Ltd. : Research Funding. Matsui:Shionogi & Co., Ltd. : Research Funding. Kanakura:Shionogi & Co., Ltd. : Research Funding. Matsumura:Shionogi & Co., Ltd. : Research Funding.

Mesenchymal Stem Cells Preconditioned with Myeloma Cells from High-Risk Patients Support the Growth of Myeloma Cells from Low-Risk Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3304-3304 ◽  
Author(s):  
Syed Mehdi ◽  
Sharmin Khan ◽  
Wen Ling ◽  
Randal S Shelton ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
High Risk ◽  
Cell Growth ◽  
Research Funding ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Myeloma Cells ◽  
Risk Patients

Abstract Introduction: Each disease stage in myeloma (MM) is associated with parallel changes in both the MM clone and the bone marrow (BM) microenvironment. Mesenchymal cell lineages derived from mesenchymal stem cells (MSCs), including osteoblasts, adipocytes and pericytes play an important role in MM cell growth mediated by the modification of the MM niche in the BM. The overall goal of the study was to test and identify changes induced in MSCs by high-risk (HR) MM cells that impact MSC function and promote oncogenic pathways capable of supporting low-risk (LR) MM cells. Methods: Normal MSCs were either cultured alone ("unconditioned") or co-cultured with MM cells for 5 days. The cultured and co-cultured cells were trypsinized, replated for 40 min, followed by serial washing to remove MM cells from the adherent MSCs. More than 95% of the remaining adherent cells after co-culture were MSCs ("preconditioned"). The unconditioned and preconditioned MSCs or their 24 hrs conditioned media (CM; 50%) were tested for their ability to support the 5-days growth of CD138+ MM cells from LR (n=4) and HR (n=3) patients. To identify factors altered in MSCs by HR MM cells, the unconditioned and preconditioned MSCs and their serum-free conditioned media (n=4) underwent gene expression profiling and proteomic analysis. Whole bone biopsy gene expression profiles from newly diagnosed patients with MM enrolled in Total Therapy clinical trials were used to correlate the altered expression of factors in preconditioned MSCs with their expression in clinical samples. Results: Growth of all MM cells tested was increased by inclusion of MSCs preconditioned with HR MM cells by 2.2± 0.2 (p<0.0004) and by CM from these MSCs by 9.6±2.0 (p<0.006), compared to culture of MM cells in fresh media. In contrast, CM from unconditioned MSCs increased growth of HR MM cells by 2.6±0.6 (p<0.01) fold but had minor effect on growth of LR MM cells. CM from MSCs preconditioned with HR MM cells increased growth of LR and HR MM cells by 5.7±0.1 (p<0.0002) and 2.6±1.2 (p<0.04), compared to culture of MM cells in CM from unconditioned MSCs, respectively. Growth of LR MM cells was higher by 2.9±0.3 fold using CM from MSCs preconditioned with HR MM cells than by using CM from MSCs preconditioned with LR MM cells (p<0.005). To determine the role of cell-cell contact, we compared the effect of the preconditioned MSCs and their CM on growth of LR and HR MM cells. Growth of LR MM cells (p< 0.003) and HR MM cells (p< 0.005) was higher when cultured in CM than in co-culture with MSCs. These data imply that soluble factors from preconditioned MSCs support MM cell proliferation and that adhesion of MM cells to MSCs may restrain proliferation. Genes overexpressed in preconditioned MSCs included growth factors (e.g. IL6) and receptors (e.g. EDNRA); genes underexpressed include factors associated with activity of osteoblasts (e.g. ITGBL1) and adipocytes (IGFBP2). A proteomic analysis showed a reduced level of the secreted factors IGFBP2 and ITGBL1 and increase level of IL6 in CM from MSCs preconditioned with HR MM cells compared to CM from unconditioned MSCs. IGFBP2 mediates local bioavailability of IGF1 and IGF2 and is also involved in bone formation and angiogenesis independently of the IGF axis. ITGBL1 is involved in osteoblastogenesis whereas EDNRA is known to be expressed by pericytes. Global gene expression profiles from patient material showed that EDNRA and IGFBP2 are not expressed in MM cells but are highly expressed in cultured MSCs compared to hematopoietic cells in buffy coat BM samples. EDNRA is overexpressed (p<0.005) whereas IGFBP2 is underexpressed (p<0.005) in whole BM biopsy samples from MM patients with HR disease compared to patients with LR disease (p<0.005) and in Focal Lesion compared to random BM biopsies taken from the same patients (p<0.0000006 for EDNRA and p<0.02 for IGFBP2). IHC staining of patients' bone biopsies showed higher numbers of EDNRA+ mesenchymal-like cells in MM (n=10) than MGUS/AMM (n=10, p<0.0003) and in HR MM BM than LR MM BM (p<0.03). IHC analysis also revealed that IGFBP2 is highly expressed by immature adipocytes and that its expression in these cells is reduced in HR MM BM. Conclusion: Preconditioning of MSCs is essential for promoting growth of MM cells from LR patients. Factors altered in MSCs by HR MM cells are linked to signaling pathways known to directly stimulate MM cell growth and markers associated with distinct MSC lineages changed in HR MM niche. Disclosures Davies: Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Barlogie:Signal Genetics: Patents & Royalties. Morgan:Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

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