Targeting Meningeoma-1 Driven AML through Epigenetic Modulation of the Cell of Origin

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 838-838
Author(s):  
Simone Riedel ◽  
Kathrin M Bernt ◽  
Jessica Haladyna ◽  
Matthew Bezzant ◽  
Brett Stevens ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Ex Vivo ◽  
Cell Of Origin ◽  
Loss Of Function ◽  
New Paradigm ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Epigenetic Modulation ◽  
High Level

Abstract Meningeoma-1 (MN1) overexpression in AML is common and predicts a poor prognosis. Forced expression of MN1 in early murine hematopoietic progenitors (CMP and LSK) but not hematopoietic stem cells (HSC) or committed progenitors (GMP) induces an aggressive myeloid leukemia as a single hit. This leukemia is strictly dependent on the high-level expression of a defined gene expression program in the cell of origin, which includes the key component HoxA9. This “susceptibility program” has been proposed as a therapeutic target in MN1high AML, but means to modulate this program have so far remained elusive. We previously showed that the pharmacologically targetable histone methyltransferase Dot1l is critically required for HoxA9 expression in MLL-rearranged leukemias. We now report dependence on Dot1l of the MN1 “susceptibility-program” in murine MN1-driven leukemia using a genetic loss of function model. Key genes including HoxA9 depend on Dot1l in normal LSK-cells, and in MN1 transduced leukemogenic early progenitors. Inactivation of Dot1l prevents leukemia establishment and abrogates leukemia maintenance in vivo. Phenotypically, deletion of Dot1l in MN1-driven leukemias induces differentiation, cell cycle arrest and apoptosis. RNA-Seq data document that deletion of Dot1l antagonizes the cell of origin-derived susceptibility program in MN1 leukemias, including HoxA9 expression. We also confirm previous reports that MN1-transduced HSC can expand ex vivo but fail to cause leukemia in vivo. This has been linked to a lower expression level of HoxA9 in normal and MN1-transformed HSCs compared to early progenitors. We confirm that HoxA9 expression levels in MN1-transduced HSCs are lower than in MN1 transduced progenitors. Importantly, in contrast to MN1-transduced progenitors, this lower, “HSC-level” expression of HoxA9 is independent of Dot1l. This suggests a developmental switch at the HSC-to-progenitor transition that involves increased expression of HoxA9 and a change from Dot1l-independent to Dot1l-dependent regulation. The high HoxA9 expression necessary to cooperate with MN1 to cause AML is strictly Dot1l-dependent. HOXA9 is co-expressed with MN1 in a subset of clinical MN1high AML. Exposure of MN1highHOXA9high AML primary patient samples to a pharmacologic inhibitor of DOT1L (EPZ4777) demonstrated sensitivity to DOT1L inhibition as expected from the murine model. Taken together, these data point to DOT1L as a potential therapeutic target in MN1high AML. The targeted epigenetic modulation of the interplay between an oncogenic lesion and its cooperating normal developmental program could represent a new paradigm for the treatment of AML. Disclosures Pollyea: Celgene: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme : Consultancy.

A GATA-2-GPR65 Regulatory Module Controls Hematopoietic Stem Cell Generation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1176-1176
Author(s):  
Xin Gao ◽  
Tongyu Wu ◽  
Jamie Lahvic ◽  
Kirby D. Johnson ◽  
Erik A. Ranheim ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Rt Pcr ◽  
Employment Equity ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
G Protein Coupled

Abstract The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. We demonstrated that a deletion of a Gata2 cis-element reduced GATA-2 levels in the AGM and abrogated the capacity of hemogenic endothelium to generate HSCs. Consistent with the defective HSC generator, the mutant fetal livers were deficient in hematopoietic stem and progenitor cells (HSPCs). Using an ex vivo intact AGM culture system, we demonstrated that retrovirus-mediated GATA-2 expression in the +9.5-/- AGM rescues its hematopoietic defect. Thus, the reduced GATA-2 levels in the +9.5-/- AGM cause the HSC generation defect, and this rescue assay provides a unique system to decipher the downstream genetic network. To discover novel druggable regulators in the GATA-2 pathway to promote HSC generation, we profiled the expression pattern of all G-protein-coupled-receptors, which represent the most successful class of pharmaceutical targets, in the AGM using our RNA-seq dataset (+9.5+/+ vs. +9.5-/- AGM). This global GPCR analysis revealed four GATA-1 and GATA-2 co-regulated genes, Adora3, Gpr65, Ltb4r1, and Adora2b. Database mining revealed that only the Gpr65 expression pattern resembled that of Gata2. To evaluate GPR65 functions during HSC generation, we conducted an shRNA-based loss-of-function analysis in the AGM. While downregulating Gpr65 did not alter the abundance of the CD31+ c-Kit+ hematopoietic cell population, it significantly increased the CD31+ c-Kit+ Sca1+ HSC-containing cell population (1.4 fold, p<0.05), indicating that GPR65 suppresses HSC generation. To validate the involvement of GPR65 during the HSC generation process in vivo, we conducted a morpholino oligonucleotide (MO)-based loss-of-function study in zebrafish. In situ hybridization analysis revealed high Runx1/c-Myb expression (labeling definitive HSCs and progenitors) in 48% of embryos injected with Gpr65 MOs compared with 11% of wild type embryos. Consistent with the ex vivo AGM analysis, this increase in Runx1/c-Myb expression upon Gpr65 MO treatment suggests GPR65 is a negative regulator of HSC emergence in vivo. To dissect the molecular mechanism governing GPR65-suppressed HSC generation, we tested whether lowering Gpr65 levels altered the expression of key HSC regulators. Quantitative RT-PCR analysis revealed that downregulating Gpr65 by 60-70% in AGM CD31+ c-Kit- endothelialcells increased Gata2 mRNA by 2.9 fold (p<0.05), Gata2 primary transcripts by 3.9 fold (p<0.05), and elevated expression of the GATA-2 target gene Runx1 (2.9 fold, p<0.05). These results support a mechanism whereby GPR65-mediated Gata2 repression is an important determinant of GPR65-suppressed HSC generation. In addition to this important function in the AGM, Gpr65 knockdown studies in primary fetal liver HSPCs revealed GPR65 suppression of Gata2 transcription to the same magnitude as in the AGM. To determine if GPR65-mediated Gata2 repression requires the +9.5 site, we infected freshly isolated HSPCs from fetal livers heterozygous for the +9.5 site with retrovirus expressing shRNA targeting Gpr65. Quantitative RT-PCR with allele-specific primers revealed that Gpr65 knockdown significantly upregulates Gata2 primary transcripts from the wild type (3.1 fold, p<0.01), but not the 9.5 mutant, allele. These results establish a requirement of the +9.5 site for GPR65 to repress Gata2 transcription. As we reported that SetD8, the only enzyme known to monomethylate H4K20, represses Gata2 expression via the +9.5 site, we tested whether GPR65 represses Gata2 expression through SetD8. H4K20me1 ChIP revealed that downregulating Gpr65 significantly reduces H4K20me1 levels at the +9.5 site by 30% (p<0.005), suggesting that GPR65 repression of Gata2 transcription involves SetD8. Our studies indicate that a G-protein coupled receptor, GPR65, is negative regulator of HSC generation and establish a GATA-2-GPR65 Type Iincoherent feedforward loop that controls HSC generation, providing a foundation to develop new targets for expanding HSCs for transplantation therapies and a new druggable target to treat hematologic disorders. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Prolonged High-Level Detection of Retrovirally Marked Hematopoietic Cells in Nonhuman Primates after Transduction of CD34+ Progenitors Using Clinically Feasible Methods.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1137-1137
Author(s):  
Tong Wu ◽  
Hyeoung Joon Kim ◽  
Stephanie E. Sellers ◽  
Kristin E. Meade ◽  
Brian A. Agricola ◽  
...  
Keyword(s):  
Stem Cell ◽  
Gene Transfer ◽  
Nonhuman Primates ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Colony Pcr ◽  
High Level

Abstract Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell(HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells(STR), or the fibronectin fragment CH-296(FN), and various cytokines such as flt3 ligand(FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor(SCF), and FLT(our best cytokine combination in prior studies)with a combination of megakaryocyte growth and development factor(MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10–15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.

scholarly journals Editing the LRF Repressor Binding Site in the γ-Globin Promoters Induces Therapeutically Relevant Fetal Hemoglobin Levels for the Treatment of β-Hemoglobinopathies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Giacomo Frati ◽  
Panagiotis Antoniou ◽  
Giulia Hardouin ◽  
Bochra Mlaya ◽  
Leslie Weber ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Ex Vivo ◽  
Fetal Hemoglobin ◽  
Human Cells ◽  
End Joining ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Cas9 Nuclease

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult β-globin chain. In β-thalassemia, the reduced (β+) or absent (β0) production of adult β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent red blood cell (RBC) sickling, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 disrupt the binding sites (BS) for transcriptional repressors (e.g., BCL11A and LRF). Recently, we demonstrated that CRISPR/Cas9-mediated disruption of the LRF BS in the HBG promoters via non-homologous end joining and microhomology-mediated end joining (MMEJ) repair mechanisms mimics the effect of HPFH mutations by impairing the LRF binding and re-activating the γ-globin expression (Weber, Frati et al., Science Advances, 2020). Efficient editing of the LRF BS (≥ 3 γ-globin promoters in &gt;70% of SCD hematopoietic stem/progenitor cells (HSPCs)) resulted in a robust HbF reactivation and a concomitant reduction in βS-globin levels recapitulating the phenotype of asymptomatic SCD-HPFH patients. RBCs derived from edited HSPCs displayed HbF levels sufficient to correct the SCD cell phenotype. Similarly, LRF BS targeting in β0-thalassemic cells results in HbF reactivation potentially correcting the α/β-like globin imbalance. Xenotransplantation of human HSPCs edited using several gRNAs targeting the LRF BS showed a robust engraftment of edited cells that were capable to differentiate into multiple lineages. HBG editing in engrafted cells ranged from 26% to 76% with a decrease (-33%) of editing events compared to the input HSPCs, partially due to a reduced occurrence of MMEJ-mediated events in hematopoietic stem cells (HSCs). Erythroid progenitors (BFU-E) obtained ex vivo from engrafted human cells, showed a relevant γ-globin expression (~40% of the total β-like chain) despite of the reduction in the number of edited promoters per BFU-E after transplantation. Moreover, mature RBCs ex vivo differentiated from edited human cells ensure therapeutically relevant HbF levels. Sequencing of top-scoring off-targets identified by GUIDE-seq showed a relatively high off-target activity within an intergenic site devoid of known regulatory elements both in vitro and in vivo in primary human cells treated with one of the gRNAs targeting the LRF BS. Although the occurrence of this off-target event in repopulating cells suggests that it has no detrimental effect on HSC engraftment and multilineage differentiation, we tested high fidelity Cas9 variants to reduce off-target activity in primary HSPCs. Finally, we used CAST-Seq assay to evaluate the potential chromosomal rearrangements in edited primary human cells in vitro and in vivo. To minimize the potential genotoxicity associated to Cas9 nuclease-mediated double-strand breaks (DSBs) in the genome, we explored the base editing system to introduce C&gt;T point mutations in the LRF BS without generating DSBs. The absence of the canonical SpyCas9 NGG PAM close to the LRF BS, prompted us to use known and novel base editors containing non-NGG Cas9 variants that allowed the editing of up to 6 out of 8 cytosines of the LRF BS in erythroid cell lines and in primary HSPCs from SCD patients. These C&gt;T conversions include not only known HPFH mutations but also mutations that can further impair LRF binding. In the majority of cases, we detected no insertions or deletions in base-edited samples, as compared to nuclease-edited samples. The 4.9-kb deletion that can be generated upon cleavage of the two identical HBG promoters by the Cas9 nuclease was barely detected in base-edited samples. Importantly, disruption of the LRF BS by non-NGG enzymes led to HbF de-repression, without affecting erythroid differentiation. This work identifies the LRF BS as an effective and safe therapeutic target for the treatment of β-hemoglobinopathies. Disclosures Casini: Alia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

scholarly journals High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells

2019 ◽  
Vol 3 (19) ◽  
pp. 2883-2894 ◽  
Author(s):  
Hongjie Wang ◽  
Zhinan Liu ◽  
Chang Li ◽  
Sucheol Gil ◽  
Thalia Papayannopoulou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Side Effects ◽  
Protein Production ◽  
Hemophilia A ◽  
Ex Vivo ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Key Points ◽  
High Level

Key Points An in vivo HSC transduction/selection allows for high-level protein expression from erythroid cells without side effects on erythropoiesis. This approach that did not require ex vivo HSC manipulation and transplantation resulted in phenotypic correction of murine hemophilia A.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals ZMYND11-MBTD1 induces leukemogenesis through hijacking NuA4/TIP60 acetyltransferase complex and a PWWP-mediated chromatin association mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Li ◽  
Phillip M. Galbo ◽  
Weida Gong ◽  
Aaron J. Storey ◽  
Yi-Hsuan Tsai ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Fusion Gene ◽  
Ex Vivo ◽  
Chromosomal Translocation ◽  
Regulatory Elements ◽  
Hematopoietic Stem ◽  
Its Gene ◽  
Human Acute Myeloid Leukemia ◽  
Bromodomain Proteins

AbstractRecurring chromosomal translocation t(10;17)(p15;q21) present in a subset of human acute myeloid leukemia (AML) patients creates an aberrant fusion gene termed ZMYND11-MBTD1 (ZM); however, its function remains undetermined. Here, we show that ZM confers primary murine hematopoietic stem/progenitor cells indefinite self-renewal capability ex vivo and causes AML in vivo. Genomics profilings reveal that ZM directly binds to and maintains high expression of pro-leukemic genes including Hoxa, Meis1, Myb, Myc and Sox4. Mechanistically, ZM recruits the NuA4/Tip60 histone acetyltransferase complex to cis-regulatory elements, sustaining an active chromatin state enriched in histone acetylation and devoid of repressive histone marks. Systematic mutagenesis of ZM demonstrates essential requirements of Tip60 interaction and an H3K36me3-binding PWWP (Pro-Trp-Trp-Pro) domain for oncogenesis. Inhibitor of histone acetylation-‘reading’ bromodomain proteins, which act downstream of ZM, is efficacious in treating ZM-induced AML. Collectively, this study demonstrates AML-causing effects of ZM, examines its gene-regulatory roles, and reports an attractive mechanism-guided therapeutic strategy.

scholarly journals LINC00680 enhances hepatocellular carcinoma stemness behavior and chemoresistance by sponging miR-568 to upregulate AKT3

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Gege Shu ◽  
Huizhao Su ◽  
Zhiqian Wang ◽  
Shihui Lai ◽  
Yan Wang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Mechanism Study ◽  
Downstream Signaling ◽  
Mouse Xenograft Model ◽  
High Level

Abstract Background Hepatocellular carcinoma (HCC) has an extremely poor prognosis due to the development of chemoresistance, coupled with inherently increased stemness properties. Long non-coding RNAs (LncRNAs) are key regulators for tumor cell stemness and chemosensitivity. Currently the relevance between LINC00680 and tumor progression was still largely unknown, with only one study showing its significance in glioblastoma. The study herein was aimed at identifying the role of LINC00680 in the regulation HCC stemness and chemosensitivity. Methods QRT-PCR was used to detect the expression of LINC00680, miR-568 and AKT3 in tissue specimen and cell lines. Gain- or loss-of function assays were applied to access the function of LINC00680 in HCC cells, including cell proliferation and stemness properties. HCC stemness and chemosensitivity were determined by sphere formation, cell viability and colony formation. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were performed to examine the interaction between LINC00680 and miR-568 as well as that between miR-568 and AKT3. A nude mouse xenograft model was established for the in vivo study. Results We found that LINC00680 was remarkably upregulated in HCC tissues. Patients with high level of LINC00680 had poorer prognosis. LINC00680 overexpression significantly enhanced HCC cell stemness and decreased in vitro and in vivo chemosensitivity to 5-fluorouracil (5-Fu), whereas LINC00680 knockdown led to opposite results. Mechanism study revealed that LINC00680 regulated HCC stemness and chemosensitivity through sponging miR-568, thereby expediting the expression of AKT3, which further activated its downstream signaling molecules, including mTOR, elF4EBP1, and p70S6K. Conclusion LINC00680 promotes HCC stemness properties and decreases chemosensitivity through sponging miR-568 to activate AKT3, suggesting that LINC00680 might be a potentially important HCC diagnosis marker and therapeutic target.

scholarly journals Systemic T Cell–independent Tumor Immunity after Transplantation of Universal Receptor–modified Bone Marrow into SCID Mice

1996 ◽  
Vol 184 (6) ◽  
pp. 2261-2270 ◽  
Author(s):  
Kristen M. Hege ◽  
Keegan S. Cooke ◽  
Mitchell H. Finer ◽  
Krisztina M. Zsebo ◽  
Margo R. Roberts
Keyword(s):  
T Cell ◽  
Ex Vivo ◽  
Lethal Dose ◽  
Cell Receptor ◽  
Scid Mice ◽  
Human Leukemia ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Hiv Envelope

Gene modification of hematopoietic stem cells (HSC) with antigen-specific, chimeric, or “universal” immune receptors (URs) is a novel but untested form of targeted immunotherapy. A human immunodeficiency virus (HIV) envelope–specific UR consisting of the extracellular domain of human CD4 linked to the ζ chain of the T cell receptor (CD4ζ) was introduced ex vivo into murine HSC by retroviral transduction. After transplantation into immunodeficient SCID mice, sustained high level expression of CD4ζ was observed in circulating myeloid and natural killer cells. CD4ζ-transplanted mice were protected from challenge with a lethal dose of a disseminated human leukemia expressing HIV envelope. These results demonstrate the ability of chimeric receptors bearing ζ-signaling domains to activate non–T cell effector populations in vivo and thereby mediate systemic immunity.

scholarly journals Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Paul D. Bates ◽  
Alexander L. Rakhmilevich ◽  
Monica M. Cho ◽  
Myriam N. Bouchlaka ◽  
Seema L. Rao ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

scholarly journals Epigenetic Activation of the pH Regulator MCT4 in Acute Myeloid Leukemia Exploits a Fundamental Metabolic Process of Enhancing Cell Growth through Proton Shifting

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3765-3765
Author(s):  
Cheuk-Him Man ◽  
David T. Scadden ◽  
Francois Mercier ◽  
Nian Liu ◽  
Wentao Dong ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Monocarboxylate Transporter ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Acute Myeloid

Acute myeloid leukemia (AML) cells exhibit metabolic alterations that may provide therapeutic targets not necessarily evident in the cancer cell genome. Among the metabolic features we noted in AML compared with normal hematopoietic stem and progenitors (HSPC) was a strikingly consistent alkaline intracellular pH (pHi). Among candidate proton regulators, monocarboxylate transporter 4 (MCT4) mRNA and protein were differentially increased in multiple human and mouse AML cell lines and primary AML cells. MCT4 is a plasma membrane H+and lactate co-transporter whose activity necessarily shifts protons extracellularly as intracellular lactate is extruded. MCT4 activity is increased when overexpressed or with increased intracellular lactate generated by glycolysis in the setting of nutrient abundance. With increased MCT4 activity, extracellular lactate and protons will increase causing extracellular acidification while alkalinizing the intracellular compartment. MCT4-knockout (MCT4-KO) of mouse and human AMLdid not induce compensatory MCT1 expression, reduced pHi, suppressed proliferation and improved animal survival. Growth reduction was experimentally defined to be due to intracellular acidification rather than lactate accumulation by independent modulation of those parameters. MCT4-KOmetabolic profiling demonstrated decreased ATP/ADP and increased NADP+/NADPH suggesting suppression of glycolysis and the pentose phosphate pathway (PPP) that was confirmed by stable isotopic carbon flux analyses. Notably,the enzymatic activity of purified gatekeeper enzymes, hexokinase 1 (HK1), pyruvate kinase M2 isoform (PKM2) and glucose-6-phosphate dehydrogenase (G6PDH) was sensitive to pH with increased activity at the leukemic pHi (pH 7.6) compared to normal pHi (pH 7.3). Evaluating MCT4 transcriptional regulation, we defined that activating histonemarks, H3K27ac and H3K4me3, were enriched at the MCT4 promoter region as were transcriptional regulators MLL1 and Brd4 by ChIP in AML compared with normal cells. Pharmacologic inhibition of Brd4 suppressed Brd4 and H3K27ac enrichment and MCT4 expression in AML and reduced leukemic cell growth. To determine whether MCT4 based pHi changes were sufficient to increase cell proliferation, we overexpressed MCT4 in normal HSPC and demonstrated in vivo increases in growth in conjunction with pHi alkalization. Some other cell types also were increased in their growth kinetics by MCT4 overexpression and pHi increase. Therefore, proton shifting may be a means by which cells respond to nutrient abundance, co-transporting lactate and protons out of the cell, increasing the activity of enzymes that enhance PPP and glycolysis for biomass generation. Epigenetic changes in AML appear to exploit that process by increasing MCT4 expression to enforce proton exclusion thereby gaining a growth advantage without dependence on signaling pathways. Inhibiting MCT4 and intracellular alkalization may diminish the ability of AML to outcompete normal hematopoiesis. Figure Disclosures Scadden: Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership.

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