scholarly journals Eltrombopag Creates a Transient Chemical Phenocopy of TET2 Loss of Function That Contributes to Hematopoietic Precursor Expansion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 453-453
Author(s):  
Yihong Guan ◽  
Bhumika J. Patel ◽  
Metis Hasipek ◽  
Dale Grabowski ◽  
Hassan Awada ◽  
...  
Keyword(s):  
Board Of Directors ◽  
In Silico ◽  
Structural Model ◽  
Data Monitoring Committee ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Free System ◽  
Dose Dependent

Eltrombopag (Epag) is FDA approved for immune thrombocytopenic purpura (ITP) and aplastic anemia (AA), in which it induces tri-lineage responses in primary and refractory settings. These biologic effects suggest that Epag helps to regenerate not only committed megakaryocytic progenitors, but also hematopoietic stem and progenitor cells (HSPCs). Epag is a small molecule thrombopoietin receptor (TpoR) agonist that activates the JAK-STAT pathway to increase platelet counts similar to the polypeptide based TpoR agonist Nplate. In addition, some of Epag's activity may, unlike that of Nplate, be independent of TpoR. Epag increases HSC self-renewal in mice despite the lack of binding to murine TpoR and showed efficacy in a TpoR-deficient strain. Here we show that Epag binds and inhibits TET2 in an iron-chelation independent manner, to in this way increase precursor expansion. Since iron is a key prosthetic component of the TET2 enzyme, we determined if Epag sequestration of iron in HSPCs inhibits TET2 function. In silico modeling indicated that Epag can form a tripartate complex with Fe2+, αKG and TET2 (Fig.A). Epag interacted with TET2 via N1387 and H1984 forming a two-way H-bond and also coordinating Fe2+ sandwiched between N-Oxalylglycine a surrogate for aKG and H1381 residues of TET2 (Fig.A). To experimentally confirm the computational structural model and study the effect of Epag on TET2, we used an ELISA-based TET2 activity assay in a cell-free system. We found that Epag inhibits TET2 in a dose-dependent manner with an IC50 of 1.6±0.1 µM in the presence of 25 µM each of aKG and Fe2+ (Fig.B). Interestingly, this observed IC50 of Epag for TET2 inhibition is 10-fold lower than the plasma Cmax of Epag that is produced in humans at standard clinical doses. Therefore, we performed a dose dependent TET2 rescue experiment by increasing aKG and Fe2+. There was no proportional effect on the TET2 inhibitory IC50 of Epag upon increasing either Fe2+ or αKG suggesting the inhibition of TET2 is independent of both these co-factors (Fig.B). This was consistent with in silico structural model data indicating that Epag specifically binds and traps TET2 in an inactive state, explaining why increasing concentration of Fe2+ or Fe3+ failed to rescue TET2 activity (Fig.C). Also consistent with this model of how Epag inhibits TET2, we did not experimentally observe any significant effect of ascorbic acid (100 µM), known to activate TET2 through maintenance of Fe3+↔Fe2+ homeostasis during TET2 catalysis. Underscoring likely relevance of TpoR independent actions of Epag, Epag treatment of K562 cells displaying undetectable levels of TpoR mRNA as well as protein, significantly reduced levels of 5hmC, while Tpo had no effects on 5hmC (Fig.D). We are currently measuring, after written informed consent on an IRB approved protocol, 5hmc levels serially in patients who are receiving Epag. In summary, we demonstrate TpoR-independent actions of Epag, its direct inhibition of TET2 activity, most likely by locking TET2 in an inactive configuration. Given the fundamental role of TET2 in promoting differentiation, this mechanism-of-action of Epag could be one pathway by which it expands HSPCs, independent of TpoR. In short, Epag creates a transient chemical phenocopy of TET2 loss of function, simultaneously having the capacity to activate JAK-STAT signaling via TpoR. These actions together can explain the clinical potency of Epag. Figure Disclosures Nazha: Abbvie: Consultancy; Tolero, Karyopharma: Honoraria; Daiichi Sankyo: Consultancy; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding. Saunthararajah:Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

scholarly journals Activation of ULK1 Kinase Mediates Clearance of Free Alpha-Globin in Human Beta-Thalassemic Erythroblasts

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 411-411
Author(s):  
Christophe Lechauve ◽  
Julia Keith ◽  
Eugene Khandros ◽  
Stephanie Fowler ◽  
Kalin Mayberry ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Protein Quality ◽  
Therapeutic Potential ◽  
Dependent Manner ◽  
Ineffective Erythropoiesis ◽  
Advisory Committees ◽  
Mtor Inhibition ◽  
Hematopoietic Stem ◽  
Rapamycin Treatment ◽  
Α Chain

Abstract β-Thalassemia is a common, frequently debilitating, inherited anemia caused by HBB gene mutations that reduce or eliminate the expression of the β-globin subunit of adult hemoglobin (HbA, α2β2). Consequently, excess free α-globin forms toxic precipitates in red blood cells (RBCs) and their precursors, leading to ineffective erythropoiesis and hemolytic anemia. Previously, we showed that free α-globin is eliminated by protein quality-control pathways, including the ubiquitin-proteasome system and autophagy (Khandros et al., Blood 2012;119:5265). In β-thalassemic mice, disruption of the Unc-51-like autophagy activating kinase gene (Ulk1) increased α-globin precipitates and worsened the pathologies of β-thalassemia. Treatment of β-thalassemic mice with rapamycin to inhibit mTOR (an ULK1 inhibitor) reduced α-globin precipitates, lessened ineffective erythropoiesis, and increased the lifespan of circulating RBCs in an Ulk1-dependent fashion. To investigate the therapeutic potential of rapamycin in human β-thalassemia, we treated erythroid precursors generated by in vitro differentiation of patient-derived CD34+ hematopoietic stem and progenitor cells. Reverse-phase high-performance liquid chromatography (HPLC) analysis of hemoglobinized erythroblasts generated from transfusion-dependent (TD, n = 5) or non-transfusion-dependent (NTD, n = 5) β-thalassemia patients revealed α-chain excesses (α-chain/β-like [β + γ + δ] chain) of approximately 40% and 15%, respectively (compared to 7 normal donors; P < 0.001). Rapamycin (10µM or 20µM) or the proteasome inhibitor MG132 (2.5µM) was added to day 13 cultures, which contained mid- to late-stage erythroblasts, and α-globin accumulation was determined by HPLC 2 days later. As expected, proteasome inhibition by MG132 raised free α-globin levels in thalassemic erythroblasts (P < 0.01) and induced cell death (P < 0.01). In contrast, rapamycin reduced free α-globin in a dose-dependent manner by 40% and 85% in TD (P < 0.0001) and NTD β-thalassemia (P < 0.001), respectively, but had no effect on erythroblasts derived from normal CD34+ cells (figure). We also observed decreases in the accumulation of autophagic markers, such as SQSTM1/p62 protein, by Western blotting. We observed no negative effects of rapamycin on the survival of patient-derived erythroblasts. Also of note, under our experimental conditions, rapamycin treatment of erythroblasts did not induce fetal hemoglobin production, as has been previously reported, thereby excluding this potential mechanism for reducing globin chain imbalances. Overall, rapamycin treatment significantly reduced the accumulation of free α-globin in TD β-thalassemia and almost fully corrected the imbalance in NTD β-thalassemia cells. Our findings identify a new drug-regulatable pathway for ameliorating β-thalassemia. Rapamycin is approved and well studied, and it has a generally manageable toxicity profile. Moreover, there are additional pharmacologic approaches to activating ULK via mTOR inhibition or other pathways. These approaches may lead to effective drug therapies for β-thalassemia, particularly NTD or intermittently TD forms of the disease. Disclosures Cappellini: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria.

scholarly journals AVID200, a Potent Trap for TGF-β Ligands Inhibits TGF-β1 Signaling in Human Myelofibrosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1791-1791 ◽  
Author(s):  
Lilian Varricchio ◽  
John Mascarenhas ◽  
Anna Rita Migliaccio ◽  
Maureen O'Connor-McCourt ◽  
Gilles Tremblay ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Type I Collagen ◽  
Normal Donor ◽  
Type I ◽  
Cell Transplant ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Myelofibrosis (MF) is caused by driver mutations which upregulate JAK/STAT signaling. The only curative treatment for MF is hematopoietic stem cell transplant. Ruxolitinib alleviates many of the symptoms in MF but does not significantly alter survival. There is, therefore, an urgent need for additional rational therapies for MF. Bone marrow fibrosis and collagen deposition are hallmarks of MF which have been attributed to megakaryocyte (MK) derived TGFβ, which also plays a role in myelo-proliferation. There are three isoforms of TGFβ (TGFβ1, β2, and β3). AVID200, which was constructed by fusing TGFβR ectodomains to IgG Fc regions, is a potent TGFβ trap with pM potency against two of the three TGFβ ligands, TGFβ1 and β3 (IC50 values of ~ 3 pM ). AVID200's IC50 for TGFβ2 is ~4,000-fold higher indicating that it has minimal activity against TGFβ2, which is desirable since TGFβ2 is a positive regulator of hematopoiesis. We explored the therapeutic potential of AVID200 by culturing MF or normal donor (ND) mononuclear cells (MNCs) first in the presence of stem cell factor and thrombopoietin (TPO) and then TPO alone in order to generate MK-enriched populations. Although the percentage of mature MKs from ND and MF MNCs was similar, the absolute number of CD41+/CD42+ MKs generated from MF MNCs was two-fold greater than ND MNCs. To determine the levels of TGFβ secreted by the MKs we screened MF and ND MNC conditioned media (CM). We observed significantly higher levels of TGFβ1 but not TGFβ2 and TGFβ3 in MF MK CM. The effects of AVID200 on MKs were then evaluated by measuring the levels of phosphorylated SMAD2. Treatment with 0.001 - 0.1 nM AVID200 decreased phosphorylation of SMAD2, suggesting that AVID200 blocks autocrine MK TGFβ signaling. The increased levels of TGFβ in MF patients promote the proliferation and deposition of collagen by mesenchymal stem cells (MSCs). Cellular proliferation of MSCs was evaluated following treatment with either recombinant TGFβ1 or ND/MF CM in the presence or absence of AVID200. In the absence of AVID200, both recombinant TGFβ1 and MK-derived CM increased the proliferation of MSCs by 1.4- and 1.6-fold respectively, which returned to basal levels with the addition of increasing concentrations of AVID200. These data indicate that AVID200 directly blocks the effect of TGFβ1 on MSCs. MF stroma is characterized by an increase in Type I collagen. We therefore examined if treatment with AVID200 interferes with the ability of TGFβ1 to induce collagen expression by MSCs. MSCs were cultured in presence of recombinant TGFβ1 alone or in combination with varying concentrations of AVID200 for 72 hours. Recombinant TGFβ1 alone induced an increase in COL1A1 mRNA expression as compared to untreated controls (p<0.01). Addition of AVID200 eliminated the TGFβ-mediated increase in COL1A1 expression in a dose dependent manner. ND and MF MK-derived CM also increased COL1A1 expression by MSCs as compared to un-treated controls (p<0.01) and that effect was eliminated by AVID200 treatment (p<0.01). We next demonstrated that TGFβ1 activated pSMAD2 in MSCs without affecting total SMAD2/3 expression and that SMAD2 phosphorylation was reduced by adding AVID200. Furthermore, AVID200 treatment decreased pSTAT3 which is associated with the ability of TGFβ to induce fibrosis. We next investigated the effect of AVID200 on MF hematopoiesis. Briefly, MNCs (which produce TGFβ) from two JAK2V617F+ MF patients were incubated with or without 50 nM of AVID200 and plated in semi-solid media. Treatment with AVID200 did not affect the overall number of colonies generated, but reduced the numbers of JAKV617F+ colonies while increasing the numbers of WT colonies: for PT1, there were 32% JAKV617F+ CFUs in untreated cultures (11 JAKV617F+/34 total colonies) versus 16% JAKV617F+ CFUs (7 JAKV617F+/42 total CFUs) in AVID200 treated cultures; for PT2 there were 100% JAKV617F+ CFUs in untreated cultures (37 JAKV617F+/37 total CFUs) versus 94% JAKV617F+ CFUs (49 JAK2V617F+/52 total CFUs) in AVID200 treated cultures. The in vivo effects of AVID200 on the development of MF in GATA1 low mice will be presented at the meeting. These data indicate that AVID200 selectively suppresses TGFβ1 signaling associated with the proliferation of MSCs and type I collagen synthesis, and depletes MF MNCs of JAK2V617F+progenitor cells. We conclude that AVID200 is a promising agent for treating MF patients which will be evaluated in a phase 1 clinical trial. Disclosures Mascarenhas: Novartis: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merck: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding. Iancu-Rubin:Incyte: Research Funding; Merck: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Summer Road: Research Funding; Merus: Research Funding; Janssen: Research Funding; Formation Biologics: Research Funding.

scholarly journals Eltrombopag: More Than Just a Thrombopoietin Receptor Agonist (TPO-RA) in Immune Thrombocytopenia (ITP)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2364-2364
Author(s):  
Anwar A. Sayed ◽  
Amna Malik ◽  
Grace Ayoola ◽  
Elisa Lucchini ◽  
Sasfia Candrianita ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Granzyme B ◽  
Immunomodulatory Effect ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Dose Dependent

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by a skewed proinflammatory T cell profile. Thrombopoietin-receptor agonists (TPO-RA) have largely replaced immunosuppressants in the management of this disorder, with some patients achieving remission after a period of treatment with TPO-RA. The potential immune modulatory role of TPO-RA has not been fully investigated. The two current TPO-RA licensed for use in ITP; Eltrombopag (Elt) and Romiplostim (Romi) act on different parts of the TPO-R and have similar response rates. However, patients can respond to one agent but not the other. Elt has been described to have a strong iron chelating effect, and hence we propose that it may have an additive immunomodulatory effect on the T cells, absent in Romi. We determined the immunomodulatory effect of Elt by assessing the proliferation and functionality of T-cell lines and primary T-cells. T cell proliferation was assessed using both CFSE proliferation assay and MTT cell viability assay. T cell phenotype and functionality were assessed by multicolor surface and intracellular flow cytometric staining. Cells were co-cultured with Elt and Romi in vitro and ex vivo with both Jurkat and DG75 cells lines as well as primary cells, respectively. Deferoxamine (DFX) was used as a positive control for iron-chelation, and human TPO was used as a positive control for TPO-RA. All treatment doses were based on their calculated therapeutic serum levels. Mann Whitney U and Kruskal-Wallis H statistical tests were applied where applicable, and a P value of less than 0.05 were considered significant. Elt significantly decreased Jurkat T cells proliferation in a dose-dependent manner compared to no treatment and Romi. DFX, an iron chelator, also decreased Jurkat T cell proliferation to comparable levels of Elt. Interestingly, this anti-proliferative effect of Elt was only observed on Jurkat T cells, but not DG75 B cell line. Ex vivo CFSE proliferation assay was performed on primary CD4 and CD8 T cells assessing the antiproliferative effect of Elt. Elt significantly reduced proliferation compared to no treatment. DFX exhibited a similar antiproliferative effect on primary T cells, however, less potent compared to Elt. Neither Romi nor TPO affected the proliferation of Jurkat cells, DG75 cells or primary T cells. The functionality of CD4 and CD8 T cells was assessed based on the capacity of T cells to produce intracellular TNFα, IFNγ and Granzyme B. Elt significantly reduced the percentages of TNFα+/IFNγ+ CD4+ and CD8+ T cells in a dose-dependent manner. This reduction was also observed, albeit to a lesser extent, when T cells were treated with DFX. Furthermore, Granzyme B expression in CD8+ T cells was significantly reduced when cells were treated Elt, compared to no treatment. Romi did not affect the frequency of CD8+ TNFα+/IFNγ+ populations nor the expression of Granzyme B in CD8+ T cells. CD4+ and CD8+ T cells did not express TPO-R on their surface. To confirm the immunomodulatory role of Elt in vivo, the terminally-differentiated effector (CD45RA+CD62L-) CD8+ T cells were assessed in 13 Elt-treated patients and 11 Romi-treated patients. Patients on Elt had significantly reduced frequency of effector CD8 T cells compared to Romi-treated patients (44% vs 76.8%; p<0.01). Taken together, these novel findings suggest an off target immunomodulatory nature of Elt besides its thrombopoietic effect. This dose-dependent immunomodulatory effect is not TPO-R dependent and targets T cells primarily. This study is the first to display such property of Elt and could explain why there is a differential response to Elt and Romi. We hypothesise that Elt may be more effective in patients with T cell mediated disease, whilst patients with predominantly antibody mediated disease are more likely respond to Romi. These findings can also offer an explanation for Elt effectiveness in other T cell-mediated autoimmune conditions such as Aplastic Anemia. Disclosures Cooper: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

A Randomized, Double-Blind, Placebo-Controlled Phase II Trial on the Efficacy, Safety and Tolerability of E5501 (AKR501) In Subjects with Chronic Immune Thrombocytopenia (ITP)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 71-71 ◽  
Author(s):  
James B. Bussel ◽  
Jenny Zhang ◽  
Shande Tang ◽  
Joe McIntosh ◽  
David J. Kuter
Keyword(s):  
Platelet Count ◽  
Board Of Directors ◽  
Research Funding ◽  
Responder Rate ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Double Blind ◽  
Baseline Platelet Count ◽  
Platelet Counts ◽  
Dose Dependent

Abstract Abstract 71 Chronic immune thrombocytopenia (ITP) is a condition of low platelet counts due to increased autoimmune-mediated platelet destruction and suboptimal platelet production. Thrombopoietin (TPO) receptor agonists are a novel class of agents which increase platelet counts by mimicking the principal physiologic regulator of platelet production, TPO. TPO agonists have demonstrated efficacy in randomized controlled trials in patients with ITP who are refractory to 1st and 2nd line agents. E5501 (previously AKR501) is a novel, orally-active, once-a-day TPO agonist which increased platelet counts in healthy volunteers. Here, we report data from a Phase II, multicenter, randomized, double-blind, placebo-controlled, dose-ranging, parallel group 4-week study (501-CL-003) of E5501 in subjects with ITP whose disease was refractory to, or had relapsed after, at least one prior ITP therapy. Subjects were enrolled if they had a baseline platelet count of <30 x109/L, or if they had a baseline platelet count of <50 x109/L and were on stable corticosteroid therapy. Sixty-four subjects were randomized to E5501 (2.5, 5, 10 or 20 mg) or placebo in a 3:3:3:3:1 randomization ratio, respectively. E5501 or placebo was administered orally, once daily for 28 days. Response to E5501 was based on weekly platelet counts. The primary endpoint was responder rate at Day 28. Responders were defined as subjects whose platelet count was ≥50 x109/L and had risen by a minimum of 20 x109/L above baseline. Responder rate increased in subjects receiving E5501 in a dose-dependent manner (Table 1). At Day 28, the responder rate in the E5501 20 mg group was 80% (12 of 15 subjects) vs 0% in the placebo group (p=0.0036). The responder rate was also significantly higher with E5501 20 mg than with E5501 2.5 mg (80% vs 13.3%; p=0.0007). In non-splenectomized subjects, the responder rate at Day 28 was 51.2% in the combined E5501 group and 88.9% in the E5501 20 mg group, compared with 44.4% and 66.7% respectively in splenectomized subjects. Median platelet counts at Day 28, and change in platelet counts above baseline, increased in subjects receiving E5501 in a dose-dependent manner (Table 2). The majority (57.6%) of subjects responded to a dose of ≥5 mg E5501 by Day 7. Subjects treated with E5501 20 mg achieved a 93.3% response rate on Day 7. None of the 5 placebo-treated subjects responded at any time during the study. E5501 was well tolerated, with a similar proportion of subjects showing treatment-emergent adverse events (TEAEs) across all dose groups. Most TEAEs were mild, transient, and resolved completely. TEAEs occurring in ≥10% of E5501-treated subjects were fatigue (20.3%), headache (20.3%) and epistaxis (15.3%). There were no clinically relevant changes in vital signs or physical examination findings. Three subjects (2 in the 2.5 mg and 1 in the 10 mg E5501 group) reported serious TEAEs. Of the two subjects in the 2.5 mg E5501 group, one reported thrombocytopenia and one reported a GI bleed; both had platelet counts <10 x109/L. The one subject in the 10 mg E5501 group, a 72-year-old Hispanic male with a significant history of cardiovascular disease (including myocardial infarction [MI], coronary artery vein bypass graft, 3 prior transient ischemic attacks [TIAs], chronic obstructive pulmonary disease, hypertension, systolic ejection murmur, angioplasty, stent placement, hyperlipidemia, and small vessel disease), had TIA and MI on Day 20 and a retinal artery occlusion 14 days after E5501 was discontinued. At the time of the events his platelet counts were 40–47 x109/L. Three other E5501-treated subjects (6.8% in total) experienced TEAEs leading to study drug withdrawal: 1 receiving 5 mg E5501 had Grade 2 musculoskeletal chest pain; 2 receiving 20 mg E5501 had excessively increased platelet counts with no clinical sequellae. In conclusion, E5501 was effective in increasing platelet counts in subjects with ITP. At 20 mg E5501, 80% of patients had responded at Day 28, with a median platelet count of 95 x109/L, and >90% of patients had responded by Day 7. E5501 was generally well tolerated and had a favorable safety profile. These data support continued development of E5501 as a potentially effective treatment with an acceptable safety profile in non-splenectomized and splenectomized patients with ITP. Disclosures: Bussel: Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Inc: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Portola: Consultancy. Zhang:Eisai: Employment. Tang:Eisai: Employment. McIntosh:Eisai: Employment. Kuter:Amgen: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Research Funding; ONO: Consultancy; Shionogi: Consultancy, Research Funding; Pfizer: Consultancy; Protalix: Consultancy, Research Funding; Risk Managment Foundation: Consultancy.

Nanoparticle Arsenic Compound Realgar Effectively Targets Myeloma Stem-Like Side Population

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4455-4455 ◽  
Author(s):  
Jana Jakubikova ◽  
Teru Hideshima ◽  
Richard W.J. Groen ◽  
Danka Cholujova ◽  
Zdenka Bujnakova ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Side Population ◽  
Dependent Manner ◽  
Tumor Initiating Cells ◽  
Advisory Committees ◽  
Ic50 Values ◽  
Anti Tumor Activity ◽  
Dose Dependent

Introduction Tumors contain diverse sub-clones, which can lead to tumor persistence and re-growth after therapy. Previous studies suggest that multiple myeloma (MM) sub-clones may contain tumor-initiating cells which maintain growth and proliferation as well as contribute to drug-resistance. Recently, we have confirmed the existence of MM stem cell-like cells by identifying “side population” (SP) cells as an enriched source of tumor-initiating cells with clonogenic and tumorigenic stem cell properties. Targeting this population with novel therapies represents a promising strategy. Arsenic trioxide (ATO, As2O3) shows only limited responses compared to anti-MM agents such as bortezomib and lenalidomide in relapsed and refractory MM. Here we evaluated the anti-MM activity of another arsenic compound (arsenic sulfide, As4S4), used in traditional Chinese medicine with greater efficacy and less genotoxicity than ATO, prepared by milling into realgar nanoparticles (NREA). We assessed the effect of NREA on MM stem cell-like SP fraction in the context of the bone marrow stromal cells (BMSCs) to provide the rational for its clinical evaluation. Methods and Results First, we evaluated the effect of both NREA and ATO on MM cell survival. Both drugs significantly decreased survival of 13 MM cell lines in a concentration-dependent manner. Importantly, we observed more potent anti-MM effect with NREA compared to ATO, with IC50 values of NREA 2-4 times lower than ATO. Moreover, we confirmed the effect of NREA on MM cells from patients with primary relapsed/refractory disease: NREA showed a dose-dependent response, with IC50 values in the range 1-2 μM. Significant concentration-dependent increased apoptosis was observed in NREA-treated MM cells. Similarly, decreased mitochondrial membrane potential was more significantly triggered by NREA than ATO. Consequently, NREA modulated the MM cell cycle profile: concentration-dependent treatment induced either G0/G1 (MM.1S and KMS11) or G2/M arrest (RPMI-S and OPM1) at 48h. To compare in vivo anti-tumor activity of NREA and ATO, we used our xenograft murine model of human MM. Tumor-bearing mice were intraperitoneally treated with NREA, ATO or with the respective vehicle. Treatment with NREA triggered more significant tumor growth inhibition compared to ATO at day 8 of treatment. To evaluate the effect of NREA on the SP phenotype of MM cells, we treated MM cells with subtoxic concentrations of NREA and ATO for 72 h. Analysis of MM cells for low intracellular content of Hoechst 33342 (SP fraction) revealed that NREA significantly decreased the percentage of SP cells in MM cells in a dose-dependent manner, whereas increase in SP fraction was observed in ATO-treated cells. To evaluate the effect of the bone marrow microenvironment on targeting the SP cells with NREA, MM cells were cultured alone or with BMSCs, and then analyzed for low intracellular accumulation of Hoechst 33342. Treatment by NREA, but not ATO, significantly depleted SP cells in co-cultures MM cells with BMSCs. Moreover, NREA decreased clonogenic potential, whereas ATO had no effect. Similarly, tumorigenic potential of SP cells was significantly decreased by NREA, but not by ATO, treatment. Conclusions Overall, our results demonstrate significant anti-tumor activity of NREA against MM cells in vitro. Our in vivo data further show that NREA significantly decreased tumor burden at clinically achievable concentrations of arsenic. Importantly, targeting of SP cells by NREA in the context of BMSCs provides the preclinical rationale for its clinical evaluation. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

scholarly journals Lessons from Nature: A Novel Class of TET Inhibitors for TET2 Mutant Associated Diseases

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4345-4345
Author(s):  
Yihong Guan ◽  
Anand D. Tiwari ◽  
Metis Hasipek ◽  
Dale Grabowski ◽  
Yvonne Parker ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Synthetic Lethality ◽  
Excision Repair ◽  
Significant Proportion ◽  
Leukemia Cell ◽  
Therapeutic Window ◽  
Loss Of Function ◽  
Advisory Committees ◽  
Free System

Abstract Discovery of many somatic lesions in leukemia enables development of targeted therapies. TET2 is one of the most frequently mutated genes in MDS/related disorders and is also present in a significant proportion of CHIP (clonal hematopoiesis of indeterminate potential) carriers. TET2 mutations (TET2mt) are mostly loss of function and occur in biallelic, heterozygous and hemi/homozygous configurations. TET2 encodes for Fe2+-dependent DNA dioxygenase that utilizes 2-ketoglutarate (αKG) for oxidation of 5-methylcytosine (mC) that results in demethylation either actively, by base excision repair of further oxidation products (fC or caC), or passively, via replication, due to DNA methyltransferase's inability to recognize 5-hydroxymethylcytosine (hmC). TET2mt are good targets for drug discovery because they often initiate the clonal evolution and are present in a large fraction of patients. However, except for ascorbic acid (AA) applied to augment TET2 activity and hypomethylating agents to which TET2mtmay be more susceptible, no specific therapies have been conceptualized for TET2mt disease. Synthetic lethality can be applied to genetic loci affected by loss of function mutations never occurring in homo/hemi/homozygous configuration. However, many tumor suppressor genes (TSG) are similar to TET2, where biallelic inactivation promotes oncogenicity and thus synthetic lethality would not be directly applicable. However, TET2 has TET1/TET3 homologs and a transient inhibition of TET enzymes could still yield a synthetic lethality (particularly in TET2mt). The idea for the proposed therapeutic strategy of TET inhibition was conceptualized based on in vivo observations of mutual exclusivity of TET2mt and IDH1/2 mutations which produces 2HG as a bone-fide natural TET inhibitor. Indeed, in our study of 485 TET2mt cases only 9 carried IDH1/2mt (mostly tiny subclones). Conversely, among 157 IDH1/2mtcases, there were only 11TET2mt (p=5.5x10-9) of which 4 were non-deleterious missense alterations or had a small clonal burden. To further support our hypothesis, we knocked in IDH1mt controlled by the doxycycline-inducible promoter into TET2mt cells. Induction of IDH1R132C or IDH1R132H (or IDH2mt), expression resulted in rapid cytotoxicity in TET2mt, and no growth perturbation was observed for TET2wt cells. Similar results have been observed in mice, where knockdown of TET3 in TET2 background shortened the life span. These observations led us to the idea of developing an aKG antagonist TET specific inhibitors (TETi). Using a structure-guided targeted discovery approach we designed, synthesized and characterized TETi, which demonstrated dose dependent inhibition of dioxygenase activity in a cell-free system. Using an iterative approach of design synthesis and activity, we selected the most potent 'hit', designated as TETi76, for further evaluation. Esterified TETi76 decreased 5hmC production and selectively induced cell death in TET2mt leukemia cell lines, SIGM5 (TET2-/-) and OCI-AML5 (TET2+/-), while a minimal effect was observed in K562 and CMK which are TET2+/+. The LD50 of TETi was 250-fold lower than 2HG in TET2mt cells. In TET2-/-engineered K562, TETi76 also showed cytotoxicity leaving a therapeutic window as compared with wild type K562. Normal bone marrows were resistant to TETi76 in clonogenic assay. In vitro mixing experiments in which Tet2mt/Tet2wt were subjected to methocult cultures at fixed ratios to mimic evolving Tet2mt clones, CD45.2 (either Tet2+/-or Tet2-/-) outcompeted CD45.1 marrow in a control setting, while treatment with TETi76 led to a gradual elimination of mutant marrow. This result was further recapitulated in vivo. In a competitive repopulation transplantation model using graft consist of a mixture of CD45 isotypes mismatched Tet2+/- or Tet2-/-and Tet2+/+marrow cells, TETi76 treatment selectively eliminated Tet2 deficient marrow. Our data have several important implications. It is likely that compensatory function from other TETs or remaining allele is needed for survival and elimination of dioxygenase appears to be lethal. This explains the exclusivity of TET2/IDH1/2 mutations. Dioxygenase inhibitors may have therapeutic applicability for selective elimination of TET2mt cells in MDS or potentially as a preventive measure in CHIP. TETi may represent a novel class of antileukemic agents. Disclosures Nazha: MEI: Consultancy. Maciejewski:Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals A Severe Mouse Model of Alpha-Thalassemia to Study Abnormal Iron Metabolism and Erythropoiesis, Hematopoietic Stem Cell Behavior and Development of a Gene Therapy Approach for Its Treatment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2012-2012
Author(s):  
Maxwell Chappell ◽  
Danuta Jadwiga Jarocha ◽  
Laura Breda ◽  
Valentina Ghiaccio ◽  
Michael Triebwasser ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Mouse Model ◽  
Board Of Directors ◽  
Globin Gene ◽  
Dependent Manner ◽  
Globin Genes ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Gene Therapy Approach ◽  
Globin Chains

Abstract Alpha thalassemia (α-thal) is caused by insufficient production of the α-globin protein because of either deletional or non-deletional inactivation of endogenous α-globin genes. Clinical presentation of deletional α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to a complete knockout (Hb Bart's Hydrops Fetalis). In patients with severe α-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation. The aims of this study are to develop a novel adult mouse model of α-thal and a gene therapy approach for this disease. We generated adult animals that do not produce α-globin chains (α-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. These animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only β-globin chains (HbH). RBC show aberrant morphology and aggregation of α- globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events. These animals show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney. Interestingly, α-KO embryos show lower numbers of FLC compared to WT embryos, lower frequency of engraftable hematopoietic stem cells (HSC; Lin-Sca-1+c-kit+CD48-), decreased clonogenic potential (fewer class 4 CFUs) and elevated erythroferrone. Lethally irradiated mice transplanted with FLC-KO require 5-6x as many cells as those transplanted with FLC-WT for recovery, further suggesting some level of engraftment impairment. Our current hypothesis is that excessive hypoxia in the embryos impairs HSC function and stem cell fitness. Additional assays are in progress to assess the nature of this impairment. To generate a gene therapy tool to rescue these animals and eventually cure severe human α-thal patients, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human α-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human α-globin genes. We identified ALS20α, a vector where α-globin is under control of the β-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous α-globin at a level comparable to that produced by one endogenous α-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Transplantation of ALS20α transduced murine BM-KO results in correction of the disease phenotype in a dose-dependent manner. At VCN&lt;1 we observe a delay in death proportional to the VCN value, while at VCN&gt;1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels. We tested ALS20α in CD34 cells isolated from four patients with both deletional and non- deletional HbH disease. We measured the change of β/α-globin mRNA ratio (β/αR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial β/αR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a β/αR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced α−thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2). In conclusion, we generated an adult mouse model of lethal α-thal and, in preliminary experiments, we rescue it with ALS20α. Furthermore, ALS20α successfully improves α-globin levels in patient cells. Further experiments are in progress to establish the consistency of our vector's expression in vivo, as well as to demonstrate its ability to transduce bona fide long-term HSCs. Disclosures Kattamis: Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Rivella: Celgene Corporation: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Dose-Dependent Role of the Cohesin Complex in Normal and Malignant Hematopoiesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 435-435
Author(s):  
Aaron D. Viny ◽  
Christopher J. Ott ◽  
Barbara Spitzer ◽  
Martin A Rivas ◽  
Cem Meydan ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Cohesin Complex ◽  
Wild Type ◽  
Advisory Committees ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Regulatory Architecture ◽  
Dose Dependent

Abstract Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability suggesting an alternate role in malignant transformation. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic stem/progenitor cells. We therefore investigated the impact of both complete loss and haploinsufficiency of Smc3, an obligate member of the cohesin complex, in normal hematopoiesis and in myeloid transformation by developing a conditional Smc3 knockout allele. Somatic loss of Smc3 in hematopoietic cells induced lethal bone marrow aplasia (median survival 11 days; p<0.001), with premature sister chromatid separation and abnormal nucleolar organization. Competitive transplant assays showed that Smc3 loss completely abrogated stem cell self-renewal in vivo. These data are consistent with an absolute requirement for the cohesin complex in hematopoietic stem/progenitor cells. By contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, with increased serial replating, expanded hematopoietic stem/progenitor cells, and a self-renewal/engraftment advantage in competitive transplantation assays in vivo (Figure a). Smc3 haploinsufficiency altered coordinated transcriptional output, including reduced expression of master regulatory transcription factors governing lineage commitment. Consistent with these data, Smc3 loss resulted in expanded Cd150+ Cd48+ ST-HSC (p=0.008), reduction in Cd150+ Cd48- LT-HSC (p=0.001), and altered chromatin architecture with dysregulated expression of genes with specific chromatin architecture footprints. Smc3 haploinsufficiency cooperated with Flt3ITD to induce acute leukemia in vivo (Figure b), with dysregulated expression of hematopoietic master regulators and altered nucleolar topology similar to that observed in germline cohesinopathy syndromes and in AML patients with cohesin mutations (Figure c). To further explore the mechanism by which Smc3 loss cooperates with Flt3ITD to induce leukemia, we investigated chromatin cis-regulatory architecture with transposase hypersensitivity assays (ATAC-seq). We hypothesized that increased accessibility at cis-regulatory elements and the alterations in gene expression seen in cells with combined Smc3 haploinsufficiency and Flt3ITD may be in a large part driven by potentiated Stat signaling at chromatin. We analyzed 146 transcription factor recognition motifs within the THS differentially observed in Smc3Δ/+Flt3ITD and wild-type cells. Chromatin accessibility gained in Smc3Δ/+Flt3ITD cells are enriched in Stat family transcription factor binding sites, including Stat5. We also observed enrichment of the Stat5 gene expression signature in the Smc3Δ/+Flt3ITD cells compared to Smc3Δ/+, Flt3ITD and wild-type cells, suggesting the divergent mutations cooperate to potentiate oncogenic Stat5 signaling in HSPCs. Our results demonstrate a key dose-dependent role for the cohesin complex in hematopoiesis, and show that reduced cohesin functions to alter enhancer-mediated transcription and contribute to aberrant self-renewal and myeloid transformation. Figure 1. Figure 1. Disclosures Levine: Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

scholarly journals The Biological Inferences from the Ranking of SF3B1 Mutations in the Clonal Hierarchy of Myeloid Neoplasia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5411-5411
Author(s):  
Hassan Awada ◽  
Jibran Durrani ◽  
Ashwin Kishtagari ◽  
Vera Adema ◽  
Cassandra M Kerr ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Chromosomal Abnormalities ◽  
Clonal Evolution ◽  
Data Monitoring Committee ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Clonal Architecture ◽  
Clonal Nature ◽  
Myeloid Neoplasia

Chromosomal abnormalities can be founder lesions (e.g., t (8; 21), inv (16), inv (3)), initiate or advance disease progression (both founder and secondary hits e.g., ASXL1, TP53, RUNX1) or can be obligatory secondary hits (FLT3, NPM1). Hence, the rank of these mutations may determine the biological properties and clinical outcomes. However, while many mechanistic studies have been undertaken without identifying the key pathogenetic factors resulting from SF3B1 mutations, important biological clues can be derived from the consequences of SF3B1 alterations in the context of the clonal architecture of myeloid neoplasia (MN). SF3B1 mutant patients often have a homogeneous phenotype with isolated erythroid dysplasia, ring sideroblasts (RS) and favorable prognoses. Studies in primary MDS cells have suggested that SF3B1 mutations are initiating lesions and provide a marked clonal advantage to MDS-RS cells by propagating from rare lympho-myeloid hematopoietic stem cells. However, there is significant diversity of clinical phenotypes and outcomes including the observation that the disappearance of RS can be observed during the disease course of clonal MN and might suggest cellular shifts due to acquisition of additional hits. In such scenarios, the cell's fate in the context of SF3B1 mutations is pre-defined by the predominance of expanded hits. We took advantage of our detailed database of molecularly and clinical annotated cases with MN to study the SF3B1 mutatome and describe whether the clonal nature (ancestral vs. secondary) might change the clinical and phenotypic trajectories of MDS cells and whether the concatenation of mutations decreases the competitiveness of SF3B1 clones, leading to the dominance of other driver genes and subsequently to clonal evolution. The clonal hierarchy was resolved using our in-house designed VAF-based bioanalytic method and confirmed by the PyClone pipeline, which showed a high level of concordance. We first assigned clonal hierarchy to SF3B1 mutations by using VAFs (adjusted for copy number and zygosity) and classifying the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent sub-clonal hit) and co-dominant hits (if the difference of VAFs between two mutations was <5%). In total, we identified 140 dominant (SF3B1DOM), 121 secondary (SF3B1SEC) and 74 co-dominant SF3B1 mutations. For the purpose of this study, we set aside co-dominant SF3B1 mutations. Focusing on SF3B1DOM and SF3B1SEC, SF3B1DOM were often associated with a normocellular bone marrow compared to SF3B1SEC (n=42 vs. 26; P=0.02) and were less likely enriched in multi-dysplastic myeloid cells (29% vs. 53%; P=0.01). As such, SF3B1DOM tended to be more frequently detected in lower-risk MDS (P=0.05) in the subtypes of MDS-RS and MLD-RS (RS≥15%: 67% vs. 41%; P=0.01) compared to other disease subtypes. Twenty-three percent of patients with SF3B1SEC had secondary acute myeloid leukemia (sAML) (P=0.03). SF3B1SEC patients tended to have a lower median platelet count than patients with SF3B1DOM (97 vs. 130 x 109/L; P=0.05). SF3B1SEC was also more associated with bi-cytopenia compared to SF3B1DOM (52% vs. 36%; P=0.01). No specific association was found between SF3B1 clonal nature and cytogenetic abnormalities, suggesting that additional mutations might be the main contributors in the evolution of MDS to AML. Of note, patients with SF3B1SEC had half OS compared to patients with SF3B1DOM (SF3B1SECvs. SF3B1DOM: 15.9 mo. vs. 39.7 mo., P= 0.0001), suggesting that in cases evolving to AML, expanding hits might have dramatically skewed the favorable nature of SF3B1 mutations. Indeed, mutations preceding SF3B1 mainly affected lineage-restricted genes associated with repression of erythroid programs (RUNX1, 23%), terminal monocytic differentiation (TET2, 9%), transcriptional corepressors (BCOR/L1, 8%) and development of leukemia (DNMT3A, 8%). In conclusion, our study of the clonal architecture of SF3B1 mutations highlights that clonal progression of cases with MN harboring SF3B1 mutations might be inferred by the rank of additional genetic lesions cooperating with SF3B1. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Abbvie: Research Funding; Macrogenics: Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Tolero, Karyopharma: Honoraria; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Abbvie: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

scholarly journals Biallelic TET2 Inactivation in Myeloid Neoplasia: From Clonal Hierarchy to Clinical Phenotypes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1805-1805
Author(s):  
Hassan Awada ◽  
Yasunobu Nagata ◽  
Abhinav Goyal ◽  
Mohammad Fahad B. Asad ◽  
Bhumika J. Patel ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Normal Karyotype ◽  
Morphological Features ◽  
Survival Outcomes ◽  
Loss Of Function ◽  
Monocytic Differentiation ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Clinical Consequences ◽  
Myeloid Neoplasia

Abstract Genomic data has led to the identification of bio-markers of morphological features and disease sub-entities in myeloid neoplasia (MN). Somatic TET2 mutations (TET2MT) are frequently found in MN, particularly in chronic myelomonocytic leukemia (CMML). TET2MT are mostly loss-of-function and hypomorphic hits leading to inactivation of TET2 protein. In fact, impaired TET2 activity skews the differentiation of hematopoietic stem cells toward proliferating myeloid precursors favoring myeloid tumorigenesis. However, the contribution of TET2MT to clinico-hematological features in MN has been controversial, possibly due to studies containing too few patients relative to the combinatorial diversity of co-occurring lesions. We recently reported on the clonal architecture of TET2MT in patients with MN. Of these, 40% of the patients harbored biallelic TET2MT (biTET2MT). Further analysis showed a frequent occurrence of biallelic TET2 inactivation (biTET2i). To date, only a few studies have investigated the clinical consequences of biTET2i in MN. We hypothesized that the presence of biTET2i identifies a group of patho-morphological features that independently define a distinct MN subtype. To test our hypothesis, we studied correlations between mutational configuration, clinico-hematological/morphological features and survival outcomes in cases that were biTET2ivs. not (biTET2-), combining whole exome and targeted deep sequencing, SNP-arrays and conventional cytogenetics. Among 1,001 clinically annotated MN patients, 82 were biTET2i (66 biTET2MT, 13 hemizygous TET2MT and 3 homozygous TET2MT, i.e. UPD) and 919 were biTET2- (96 monoallelicTET2MT and 823 wild type). TET2 hits were ancestral lesions in 72% of biTET2ivs. 38% in biTET2- cases (P<.0001). When the 1stTET2 hit was ancestral in biTET2i, the most common subsequent hit was a 2ndTET2MT, followed by SRSF2MT, ASXL1MT, KRASMT/NRASMT and DNMT3AMT. Truncation mutations (frameshift or nonsense variants) were found in 83% of biTET2ivs. 65% of biTET2- cases (P=.02). A second TET2 hit in biTET2MT cases significantly increases the accrual of additional truncating changes. Furthermore, biTET2i were significantly enriched for additional hits in SRSF2MT (33%; P<.0001) and KRASMT/NRASMT (16%; P=.03) while biTET2- for TP53MT (11%; P=.03). SRSF2MT was also found to be significantly associated with biTET2i when compared to monoallelicTET2MT (P=.02). In contrast, biTET2i cases showed absence of SRSF2MT in the absence of monocytosis. We then assessed associations of biTET2i with specific genotype/phenotype. Clinical analyses revealed that cases with biTET2i compared to cases with biTET2- were older (91% ≥60 years vs. 74%, P=.0004) and more commonly had normal karyotype (65% vs. 45%; P=.0007). BiTET2i were enriched in patients with CMML1/2 (44% vs. 9%; P<.0001), and predominantly in lower-risk cases (62% vs. 47% in biTET2-; P=.003). While a second TET2 hit occurred frequently, biTET2i did not portend faster progression but rather associated with monocytic differentiation, consistent with its prevalence in CMML. In addition, among biTET2i with SRSF2MT or KRASMT/NRASMT, CMML was diagnosed in 70% (P=.001) and 77% (P=.01) of the cases, respectively, significantly higher than what was seen in the biTET2i population (44%). In biTET2- cases, leukopenia (81%; P<.0001), neutropenia (52%; P=.008), pancytopenia (27%; P=.008) and increased marrow blast percentages (≥5% in 33%; P=.01) were more prevalent than in biTET2i cases, which in return co-segregated with monocytosis (84%; P<.0001), marrow hypercellularity (cellularity >70% in 67%; P<.0001) and marked myeloid dysplasia (68%; P=.0003). Given our observation of a highly significant (P<.0001) relationship between biTET2i, CMML diagnosis and/or monocytosis, we also evaluated patients without frank diagnosis of CMML (CMML-) and compared biTET2ivs.biTET2- for associations with monocytosis and myeloid dysplasia, two hallmarks of CMML. Increased monocyte counts among CMML-cases were significantly overrepresented in biTET2i cases (72%; P=.03) vs.biTET2- (55%) as was myeloid dysplasia (72% vs. 46%; P=.0001). Lastly, biTET2i as a sole hit or in combination with other hits did not influence survival outcomes. In sum, biTET2i invariantly associates with distinct morphological and clinical phenotype. It may thus represent an early diagnostic marker of morphologic MN sub-entities. Disclosures Nazha: MEI: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy.

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