scholarly journals Dual Targeting of Stromal Cell Support and Leukemic Cell Growth by a Peptidic PKC Inhibitor Shows Effectiveness against B-ALL

2020 ◽  
Vol 21 (10) ◽  
pp. 3705
Author(s):  
Paola Fernanda Ruiz-Aparicio ◽  
Natalia-Del Pilar Vanegas ◽  
Gloria Inés Uribe ◽  
Paola Ortiz-Montero ◽  
Camila Cadavid-Cortés ◽  
...  
Keyword(s):  
Cell Growth ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Leukemic Cell Lines ◽  
Kinase C ◽  
Dual Targeting ◽  
Pkc Isoforms ◽  
New Strategy ◽  
Intercellular Communications ◽  
Cell Support

Mesenchymal stem cells (MSC) favour a scenario where leukemic cells survive. The protein kinase C (PKC) is essential to confer MSC support to leukemic cells and may be responsible for the intrinsic leukemic cell growth. Here we have evaluated the capacity of a chimeric peptide (HKPS), directed against classical PKC isoforms, to inhibit leukemic cell growth. HKPS was able to strongly inhibit viability of different leukemic cell lines, while control HK and PS peptides had no effect. Further testing showed that 30% of primary samples from paediatric B-cell acute lymphoblastic leukaemia (B-ALL) were also strongly affected by HKPS. We showed that HKPS disrupted the supportive effect of MSC that promote leukemic cell survival. Interestingly, ICAM-1 and VLA-5 expression increased in MSC during the co-cultures with B-ALL cells, and we found that HKPS inhibited the interaction between MSC and B-ALL cells due to a reduction in the expression of these adhesion molecules. Of note, the susceptibility of B-ALL cells to dexamethasone increased when MSC were treated with HKPS. These results show the relevance of these molecular interactions in the leukemic niche. The use of HKPS may be a new strategy to disrupt intercellular communications, increasing susceptibility to therapy, and at the same time, directly affecting the growth of PKC-dependent leukemic cells.

scholarly journals Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 49-54
Author(s):  
H Kodama ◽  
M Iizuka ◽  
T Tomiyama ◽  
K Yoshida ◽  
M Seki ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
Hematopoietic Factors ◽  
In Vitro Cell Lines

Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

scholarly journals Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 49-54 ◽  
Author(s):  
H Kodama ◽  
M Iizuka ◽  
T Tomiyama ◽  
K Yoshida ◽  
M Seki ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
Hematopoietic Factors ◽  
In Vitro Cell Lines

Abstract Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

scholarly journals Single and Dual Targeting Chimeric Antigen Receptor T-Cell Therapy in Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-25
Author(s):  
Sneha Chitre ◽  
Joop Gaken ◽  
Andrea Venuso ◽  
Ghulam J. Mufti
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Target Cells ◽  
High Affinity ◽  
Leukemic Cell Lines ◽  
Dual Targeting ◽  
Car T

BACKGROUND: Discovery of chimeric antigen receptors (CARs) specific for tumour-associated antigens are emerging to be an effective form of immunotherapy for cancer treatment in recent years. However, the lack of a compelling acute myeloid leukemia (AML)-specific cell surface antigen and the safety concerns for myeloid-directed CAR T therapies causing prolonged myeloablation/aplasia which necessitate bone marrow transplantation make it challenging to develop CARTs for AML. In spite of these challenges, the high relapse rate of the disease i.e. 43% and 18% patients never attaining clinical remission (CR) with front-line induction treatments highlight an unmet need for developing improved CAR T vectors with enhanced specificity towards leukemic blasts in refractory/resistant cases with poor cytogenetics. Therefore, we aim to develop improved CAR T vectors for AML that produce safe and consistent responses in patients with high-risk disease. Herein, we demonstrate the in-vitro data for (a) efficacy of a second-generation CAR expressing six single-chain variable fragments (scFv) with different affinities for CD123 [interleukin three receptor alpha (IL3RA; CD123), a molecule over expressed on AML blasts and leukemic stem cells (LSC)] and (b) evaluate the cytotoxic effects of a dual targeting CARCD123/CD33 (against CD123 and CD33; an important myeloid marker specifically expressed on bulk AML disease) to enhance specificity towards leukemic cells therefore reducing "on-target off-organ effects". METHOD: Six lentiviral vectors with CAR against CD123 were constructed i.e. two high affinity (4nM kD & 4nM kD with a point mutation resulting in amino acid change K136Q), two moderate (56nM kD & 56nM kD with mutation at A105G) and two low affinity vectors (101nM kD & 101nM kD with mutation at V24G). To improve the specificity of the single targeting CARCD123, the high affinity (4nM kD, K136Q) vector was utilized to generate two dual targeting (CARCD123/CD33) constructs containing the activation domain (CD3ζ) directed against CD33 and the costimulatory domain (either CD28 or 41BB) directed against CD123. All constructs were transduced (MOI 1:5) into peripheral blood mononuclear cells (PBMCs) from healthy donors or AML patients and their cytotoxicity was examined by flowcytometry on leukemic cell lines; Kg1, U937, K562 [Fig:1a], Ramos wild type (CD19+, CD123-), artificially engineered Ramos cells (transduced by lentiviral vectors with CD123 and/or CD33 cDNA) i.e. Ramos 123 (CD123+), Ramos 33 (CD33+), Ramos 123/33 (CD123+ and CD33+) and AML mononucelar cells (MNCs). RESULTS: Flowcytometric analysis confirmed the expansion of T cells from PBMCs and cytotoxicity of the eight CAR constructs against target cells in in-vitro co-culture assay. High affinity CARCD123 (4nM kD & 4nM kD K136Q) T cells demonstrated enhanced cytotoxicity [Fig 1a] compared to moderate (56nM kD, 56nM kD A105G) CARCD123 in all leukemic cell lines while the low affinity (101nM kD, 101nM kD V24G) vectors had no effect. Efficacy of the high affinity CARCD123 constructs was validated on Kg1 [Fig 1b] and Ramos 123+ target cells by the increasing effector: target ratios (1:2, 1:4 & 1:10). Similar cytotoxic effects were also consistently observed against autologous AML MNCs (target cells) [n=4] and allogenic (effector cells are PBMCs from healthy donors) AML MNCs [Fig 1c] [n=3]. T cell activation was confirmed by ELISA and showed increased IFN-γ (500-2000 fold) and TNF-α (150-200 fold) levels after 24hr co-culture. Furthermore, we also elucidated the exclusive cytotoxicity of the two dual targeting CARCD123/CD33 in Ramos 123+/33+ cells [Fig 1d] with no effect being observed on Ramos 123+, Ramos 33+ and Ramos wild type (123-/33-) cell lines. This confirmed the absence of non-specific targeting, validated the improved specificity of the CARs towards leukemic cells and demonstrated a potential to reduce deleterious "on-target but off organ effects". CONCLUSION: In summary, we illustrate in-vitro data establishing the importance of scFv on CAR T cell cytotoxicity and exemplify for the first time an improved specificity of CARTs by targeting two antigens simultaneously in AML. Future work will involve examining the in-vivo dynamics of CAR CD123 and CAR CD123 CD33 on the hematopoietic system and on disease pathogenesis with an aim to proceed to phase I clinical trial. Figure 1 Disclosures Mufti: BMS, Novartis: Research Funding; Abbvie, Novartis: Consultancy.

scholarly journals Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription and leukemic cell growth

2021 ◽  
Vol 118 (6) ◽  
pp. e1922864118 ◽  
Author(s):  
Yu-Ling Lee ◽  
Keiichi Ito ◽  
Wen-Chieh Pi ◽  
I-Hsuan Lin ◽  
Chi-Shuen Chu ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Critical Role ◽  
Gene Activation ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Specific Gene ◽  
Aberrant Expression ◽  
Transcriptional Machinery

The chimeric transcription factor E2A-PBX1, containing the N-terminal activation domains of E2A fused to the C-terminal DNA-binding domain of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). We recently have reported a mechanism for RUNX1-dependent recruitment of E2A-PBX1 to chromatin in pre-B leukemic cells; but the subsequent E2A-PBX1 functions through various coactivators and the general transcriptional machinery remain unclear. The Mediator complex plays a critical role in cell-specific gene activation by serving as a key coactivator for gene-specific transcription factors that facilitates their function through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 target genes remains largely unexplored. Here we show that Mediator interacts directly with E2A-PBX1 through an interaction of the MED1 subunit with an E2A activation domain. Results of MED1 depletion by CRISPR/Cas9 further indicate that MED1 is specifically required for E2A-PBX1–dependent gene activation and leukemic cell growth. Integrated transcriptome and cistrome analyses identify pre-B cell receptor and cell cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. Notably, complementary biochemical analyses also demonstrate that recruitment of E2A-PBX1 to a target DNA template involves a direct interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1–driven leukemia. The MED1 dependency for E2A-PBX1–mediated gene activation and leukemogenesis may provide a potential therapeutic opportunity by targeting MED1 in E2A-PBX1+ pre-B leukemia.

Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1427-1434 ◽  
Author(s):  
Ivan N. Rich ◽  
Diana Worthington-White ◽  
Oliver A. Garden ◽  
Philip Musk
Keyword(s):  
Cell Cycle ◽  
Intracellular Ph ◽  
Annexin V ◽  
Leukemic Cell ◽  
Differential Sensitivity ◽  
Leukemic Cells ◽  
Hematopoietic Tissue ◽  
Leukemic Cell Lines ◽  
Targeted Inhibition

The Na+/H+ exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pHi). It is a ubiquitous, amiloride-sensitive, growth factor–activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pHi than normal hematopoietic tissue. We then show that a direct correlation exists between pHi and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na+/H+ exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pHiand induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pHi decreases, apoptosis—measured by annexin-V and TUNEL methodologies—rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents.

Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
G Manfioletti ◽  
V Gattei ◽  
E Buratti ◽  
A Rustighi ◽  
A De Iuliis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Maturation Stage ◽  
Lymphoid Tissues ◽  
Specific Cell ◽  
B Cell Differentiation ◽  
Leukemic Cell Lines

Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.

scholarly journals Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton- insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3783-3794 ◽  
Author(s):  
I Parolini ◽  
M Sargiacomo ◽  
MP Lisanti ◽  
C Peschle
Keyword(s):  
Signal Transduction ◽  
G Proteins ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Leukemic Cell ◽  
Receptor Activation ◽  
Leukemic Cells ◽  
Human Leukemic Cell ◽  
Leukemic Cell Lines ◽  
B Lymphoid

Src-family nonreceptor protein tyrosine kinases (NRPTK) are associated with cell surface receptors in large detergent-resistant complexes: in epithelial cells, yes is selectively located in vesicle structures containing caveolin (“caveolae”). These formations are typically also endowed with glycophosphatidylinositol (GPI)-anchored proteins. In the present study, we observed lck, lyn, src, hck, CD4, CD45, G proteins, and CD55 (decay-accelerating factor) expression in the buoyant low- density Triton-insoluble (LDTI) fraction of selected leukemic cell lines and granulocytes. We provide a detailed analysis of the two most highly expressed NRPTK, p53/p56lyn and p56lck, which are involved in the transduction of signals for proliferation and differentiation of monocytes/B lymphocytes and T lymphocytes, respectively. We show that lyn is selectively recovered in LDTI complexes isolated from human leukemic cell lines (promyelocytic [HL-60], erythroid [K562] and B- lymphoid [697]) and from normal human granulocytes, and that lck is recovered from LDTI fractions of leukemic T- and B-lymphoid cell lines (CEM, 697). In LDTI fractions of leukemic cells, lck and lyn are enriched 100-fold as compared with the total cell lysates. Analysis of these fractions by electron microscopy shows the presence of 70- to 200- nm vesicles: lyn and lck are homogenously distributed in the vesicles, as revealed by an immunogold labeling procedure. These novel results propose a role for these vesicles in signal transduction mechanisms of normal and neoplastic hematopoietic cells. In support of this hypothesis, we further observed that molecules participating in B- and T-cell receptor activation cofractionate in the LDTI fractions, CD45/lyn (B cells) and CD45/lck/CD4 (T cells).

scholarly journals Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 100-106 ◽  
Author(s):  
HN Steinberg ◽  
AS Tsiftsoglou ◽  
SH Robinson
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Cell Phenotype ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Leukemic Cell Lines

Abstract The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.

Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hsiangyu Hu ◽  
Nirmalya Saha ◽  
Yuting Yang ◽  
Sierrah Marie Grigsby ◽  
Rolf Marschalek ◽  
...  
Keyword(s):  
Cell Growth ◽  
Acute Leukemia ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Fusion Partner ◽  
Wild Type

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

Deletion of Bcl11a Gene Reveals Roles in Lymphoid, Myeloid, and Leukemic Cell Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 385-385 ◽  
Author(s):  
Mariaestela Ortiz ◽  
P. Liu ◽  
L. Tessarollo ◽  
R.A. Rachel ◽  
T. Nakamura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Cell Growth ◽  
B Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Cell Maturation ◽  
Lymphoid Development ◽  
Myeloid Progenitor ◽  
Human B Cell

Abstract B-cell lymphoma/leukemia 11a (Bcl11a) gene, encodes a zinc finger protein that is expressed in normal B- and T-lymphocytes, monocytes, megakaryocytes, and physically interacts with the human B-cell proto-oncogene, BCL6. Bcl11a is activated by fusion of IG the locus in human B-cell lymphomas carrying the (2;14)(p13:q32.3) translocation. To determine the function of Bcl11a in normal hematopoiesis and proliferation of leukemic cells, we generated a Bcl11a knockout mouse model. This showed that Bcl11a is essential for postnatal development and normal lymphopoiesis. While Bcl11a+/− mice are fertile, Bcl11a−/− die a few hours after birth from unknown causes. To evaluate the role of Bcl11a in hematopoiesis, fetal liver (FL) cells were analyzed by FACS for the expression of lineage specific cell surface markers. B-cell maturation was blocked prior to the development of pro-B cells (B220+/CD43+), while myeloid and erythroid lineages were normal. The Bcl11a−/− FL also showed defects in T-cell maturation (reduced CD4+ CD8− and increased CD4− CD8+ fetal thymocytes). Lethally irradiated mice transplanted with Bcl11a−/− FL cells showed normal myeloid and erythroid development, while B- and T-cell development was blocked. Thus, defects in lymphoid development were intrinsic to the hematopoietic stem cells (HSC). We established that this defect in lymphopoiesis was not due to homing to the thymus, as shown by failure of T-cell repopulation when Bcl11a−/− FL cells were injected into the thymus. Greater than 80% of the mice transplanted with Bcl11a−/− FL cells died between 15 and 27 weeks, while 100% mice transplanted with Bcl11a+/− survived. Histopathology of moribund mice showed enlarged thymus with lymphoblasts infiltrating the bone marrow, liver, lung, heart, and kidney. Furthermore, analysis by real time-PCR of thymocytes from the sick mice showed increased expression of Notch1, a negative regulator of lymphoid development. Leukemic cells were tumorogenic when injected into SCID-NOD mice, and were immortalized by co-culturing on OP9 stromal cells. Immortalized leukemic cell lines showed high expression of T-cell markers CD3e, CD4, CD8, and B-cell marker CD43. Finally, myeloid progenitor cell growth (CFU-c, 3H-Thymidine incorporation, and viable cell growth) was inhibited by over-expression of Bcl11a in bone marrow cells from 5-FU treated mice. Thus, these data show that Bcl11a is required for B and T cell differentiation and may also function as a T-cell tumor suppressor gene. Furthermore, these results are the first indication of a possible suppressive role for Bcl11a during normal myeloid progenitor development and differentiation. This project has been funded in part from NCI, NIH Contract # NO1-CO-56000, with SAIC-Fredrick, Inc.

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