CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4174-4174
Author(s):  
Jiaying Tan ◽  
Jay L. Hess
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Polycomb Group ◽  
Human Leukemia ◽  
Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

Upregulation of HLA-G Gene Expression in Malignant Hematopoietic Cells Treated with 5-Aza-2′- Ddeoxycytidine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4567-4567
Author(s):  
Firas Alsabty ◽  
Martin Mistrik ◽  
Katarina Polakova
Keyword(s):  
Gene Expression ◽  
G Protein ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Dna Demethylation ◽  
Lymphocytic Leukemia ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Demethylating Agent

Abstract Abstract 4567 Introduction Human leukocyte antigen G (HLA-G) is a nonclassic HLA class I antigen with restricted distribution in normal tissues. It exerts multiple immunregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Ectopic HLA-G expression observed in some pathological conditions such as malignant transformation may be triggered by epigenetic modifications such as DNA demethylation or histone acetylation. Materials and Methods Mononuclear cells were isolated from peripheral blood of newly diagnosed previously untreated patients with acute myeloblastic leukemia (AML) (n=9) and chronic lymphocytic leukemia (CLL) (n=5) by standard Ficoll-Hypaque density gradient centrifugation. Isolated cells were resuspended in RPMI 1640 medium supplemented with 2mM L-glutamine, 200 μg/ml gentamicin, 0.125 μg/ml amphotericin B and 10% heat-inactivated fetal bovine serum. Demethylating treatment of cells was carried out with100 μM 5-aza-2x- deoxycytidine (AdC) (Sigma) for 3 days. Human choriocarcinoma cell lines JEG3 and JAR (ATCC, Rockville, MD) were used as HLA-G positive and negative controls, respectively. Real time polymerase chain reaction (RT-PCR) and semiquantitative RT-PCR were performed using the ABI Prism 7000 Sequence Detection System and AmpliTaq Gold DNA polymerase to detect HLA-G mRNAs transcriptions. The HLA-G protein expression was examined by western blot analysis using mAb 4H84. Results HLA-G transcripts in AdC untreated leukemia samples were demonstrated in 3 out of 5 patients (60%) with B-CLL and in 5 out of 9 patients (56%) with AML. Treatment with demethylating agent AdC resulted in up-regulation of HLA-G transcription and expression of HLA-G protein in 5 out of 8 (63%) examined leukemia cell lines (Table 1). Conclusions we conclude that DNA methylation is an important control mechanism of HLA-G gene expression, and treatment of human leukemia with demethylating agent AdC may up-regulate HLA-G gene expression and induce HLA-G protein synthesis in some patients that may allow leukemic cells to escape recognition and destruction by cytotoxic T-cells or NK cells. Therefore patients should be monitored for HLA-G expression in order to follow risk of AdC therapy. Disclosures: No relevant conflicts of interest to declare.

Effect of sodium dichloroacetate on apoptotic gene expression in human leukemia cell lines

2019 ◽  
Vol 71 (2) ◽  
pp. 248-256 ◽  
Author(s):  
Jagoda Abramek ◽  
Jacek Bogucki ◽  
Marta Ziaja-Sołtys ◽  
Andrzej Stępniewski ◽  
Anna Bogucka-Kocka
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Human Leukemia Cell ◽  
Apoptotic Gene ◽  
Leukemia Cell Lines

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.

scholarly journals Proton Sensor GPR68 Is Essential to Maintain Myeloid Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1353-1353
Author(s):  
Xiaofei He ◽  
Mark Wunderlich ◽  
Benjamin Mizukawa ◽  
James C. Mulloy ◽  
Saran Feng ◽  
...  
Keyword(s):  
Cell Growth ◽  
Molecular Mechanism ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Single Case ◽  
Leukemia Cell ◽  
Disease Free Survival ◽  
Myeloid Malignancies ◽  
Extracellular Acidosis ◽  
Induced Apoptosis

Abstract Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML. Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function. In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway. In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. Disclosures No relevant conflicts of interest to declare.

mTOR Inhibitor Rapamycin Interacts Synergistically with Farnesyltransferase Inhibitor FTI-277 To Induce Growth Inhibition in Human Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1821-1821 ◽  
Author(s):  
June-Won Cheong ◽  
Ju In Eom Eom ◽  
Hye Won Lee ◽  
In-Hae Park ◽  
Yuri Kim ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Farnesyltransferase Inhibitor ◽  
Human Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Primary Leukemia

Abstract Proteins regulating the mammalian target of rapamycin (mTOR), as well as some of the targets of the mTOR kinase, are overexpressed or mutated in cancer. Rapamycin inhibits the growth of cell lines derived from multiple tumor types in vitro, and tumor models in vivo. However, it has been suggested that substantial proportion of acute myeloid leukemia (AML) cells showed resistance to rapamycin-induced growth inhibition. Aim: We aim to investigate the effects of the farnesyltransferase inhibitor (FTI)-277 on the rapamycin-induced growth inhibition of human leukemia cells. Patients and methods: Flow cytometric evaluation and Western blot analysis for mTOR and Ras-like GTPase Rheb expression in the leukemia cell lines (HL60,NB4,THP1,KG1,U937) and primary leukemia cells obtained from AML patients were performed. We also observed the inhibition of cell growth and the changes in expression of mTOR and up- or down-streams of mTOR after mTOR inhibitor rapamycin treatment with or without FTI-277. Results: Both flow cytometric evaluation and Western blot analysis demonstrated that mTOR expression in the leukemia cell lines (HL60, NB4, THP1, KG1, U937) and primary leukemia cells obtained from AML patients were significantly higher compared to normal bone marrow mononuclear cells (p<0.001). Expression of Ras-like GTPase Rheb, a mTOR upstream, was also significantly increased in the leukemia cell lines and primary AML cells compared to normal bone marrow mononuclear (p<0.001 and p<0.005, respectively). We observed the inhibition rate of leukemia cell growth after treatment of cells with mTOR inhibitor rapamycin (100mM) in the absence or presence of farnesyltransferase inhibitor FTI-277 (10mM). Clonogenic cell growth in the leukemia cell lines was 69.3 ± 5.3% in the rapamycin group and 78.7 ± 4.4% in the FTI-277 group compared to that of the control group. Cotreatment of THP1 and HL-60 leukemia cells with rapamycin and FTI-277 exerted synergistic decrease in the clonogenic cell growth, as well as arrest at the G2/M phase of cell cycle, in a dose-and time-dependent manner (p<0.01). This was associated with marked attenuation of protein levels of Rheb, phospho-mTOR, and mTOR downstreams phospho-p70S6 kinase, phospho-4E-BP1. Interestingly decreased expression of mTOR upstreams Akt/PKB activity, Akt/PKB phosphorylation and PTEN phosphorylation was also observed in these leukemia cells after cotreatment with FTI-277 and rapamycin. These findings were also observed in the primary leukemia cells obtained from untreated patients with AML. Conclusions: Taken together, these findings indicate that farnesyltransferase inhibitor FTI-277 potentially enhance the growth-inhibitory property of rapamycin, with inducing multiple perturbations in PI3K - Akt/PKB - mTOR signaling pathway in human leukemia cells. Combined rapamycin and FTI blockade can exert powerful anti-leukemia effects and could be developed into a novel therapeutic strategy for AML.

Epigenetic modulation of gene expression of human leukemia cell lines – induction of cell death and senescence

Neoplasma ◽  
2011 ◽  
Vol 58 (1) ◽  
pp. 35-44 ◽  
Author(s):  
K. ELKNEROVA ◽  
D. MYSLIVCOVA ◽  
Z. LACINOVA ◽  
I. MARINOV ◽  
L. UHERKOVA ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Human Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Modulation Of Gene Expression ◽  
Epigenetic Modulation

The Polycomb Group Transcriptional Repressor Bmi-1 Is Constitutively Expressed in Multiple Myeloma (MM) Cells and Modulates Proliferation through a Mechanism Independent of the Ink4a/ARF Locus.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3346-3346
Author(s):  
Frank J. Desarnaud ◽  
Masood Shammas ◽  
Paola Neri ◽  
Hemanta Koley ◽  
Rao Prabhala ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Critical Role ◽  
Polycomb Group ◽  
Cytokine Signaling ◽  
Polycomb Group Protein ◽  
Hematopoietic Stem

Abstract The polycomb group protein Bmi-1 is required for self-renewal of adult hematopoietic stem cells by inhibiting cell senescence and apoptosis through repression of p16INK4a and p14/p19ARF expression. Based on gene expression profile studies showing absence of Bmi-1 expression in normal plasma cells versus upregulation in all MM cell lines and primary patient MM cells, we evaluated its role in MM pathobiology. Bmi-1 expression in MM cell lines is constitutive, and is not modulated by IL-6 and IGF-1. Immunoprecipitation and immunoblotting studies confirmed both Bmi-1 overexpression and nuclear localization. Bmi-1 was found to be tyrosine- and serine-phosphorylated. We next inhibited Bmi-1 expression in MM cell lines (ARP and MM.1S) by stably transfecting a PU6 vector expressing siRNA targeting Bmi-1. Following G418 selection, cell growth, as measured by H3 thymidine incorporation and MTT assay, was stimulated 1.4 fold in MM.1S and 2 fold in ARP cells compared to control cells transfected with a PU6 vector. Gene expression profiling showed: up-regulation of IL-6 and IGF-1 receptors, as well as Gab-1, which plays a critical role in MM cell proliferation and survival induced by IL-6; Phospholipase D1 and proteinase 3 which potentiate cell proliferation by repression and cleavage of p21cip1/waf1; stem cell growth factor, HOXA9, which modulate bone marrow stem cell survival and proliferation. In contrast, p21cip1/waf1 and GADD45a, GADD45b which moderate cell growth arrest were down regulated. However, no change in expression of either p16INK4a or p14/p19ARF was detected by microarray and immunoblotting. We therefore conclude that Bmi-1 is abnormally expressed in MM cells and contrary to its classic action, modulates MM cell growth and survival by modulating cytokine signaling rather than INK4a/ARF. Our studies further suggest that Bmi-1 represents a promising target for novel MM therapeutics.

LAMP5 - a Novel Target of MLL-Fusion Proteins Is Required for the Propagation of Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1512-1512
Author(s):  
Gabriel Gracia-Maldonado ◽  
Jason Clark ◽  
James C. Mulloy ◽  
Ashish R Kumar
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Human Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Novel Target

Abstract Acute leukemias with Mixed Lineage Leukemia gene (MLL, also called KMT2A) translocations are associated with poor outcomes. These leukemias are most frequently encountered in infants and as secondary malignancies, and can be either lymphoid or myeloid. In spite of aggressive treatments, including bone marrow transplantation, infants with MLL-leukemias face a grim prognosis, with predicted survival of only 20%. Novel, effective therapies are thus urgently needed. In search for molecular targets, we observed that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5, also known as C20orf103). This observation was consistent across several gene-expression-profiling studies and occurred in both ALL and AML. Moreover, data from the TCGA study on AML showed that patients with LAMP5 expression suffered worse prognosis compared to those lacking LAMP5. We first confirmed LAMP5 expression in human leukemia cell lines by immunoblot and RT-qPCR assays. We readily detected LAMP5 mRNA and protein in MLL-fusion leukemia cell lines (MV4;11, MOLM13, THP1, RS4;11, KOPN8), while no expression of LAMP5 was found in the non-MLL-cell lines (Kasumi, K562, REH). Published ChIP-seq studies on leukemia cell lines show the MLL-fusion protein directly bound at the promotor region of LAMP5. To validate that the MLL-fusion protein activates the expression of LAMP5, we transformed human CD34+ cord blood cells with an inducible (Tet-off) retrovirus carrying the MLL-AF9 fusion cDNA. In this system, addition of Doxycycline represses expression of the MLL-AF9 oncogene. We found that LAMP5 expression directly correlated with MLL-AF9 levels, with levels of both decreasing upon addition of Doxycycline. To investigate the role of LAMP5 in MLL-fusion leukemia, we studied the effect of shRNA-mediated knockdown. By screening several hairpin sequences, we identified one construct that efficiently inhibited LAMP5 expression in MLL-fusion leukemia cells but had no effect on LAMP5-negative cells, implying specificity. All MLL-fusion leukemia cell lines tested showed growth inhibition with LAMP5 knockdown. Specifically, growth of MV4;11, THP1 and MOLM13 cells was decreased by 69%, 73% and 80% respectively compared to controls (non-targeting shRNA). When cultured in semi-solid methylcellulose media for 10 days, LAMP-5-depleted MV4;11 cells formed significantly fewer colonies than control cells (64.3 ± 25.98 and 245.3 ± 27.42 colonies per 1000 cells respectively). To investigate the role of LAMP5 in leukemia-propagation in vivo, we transplanted control and LAMP5-depleted MV4;11 cells into Busulfan-conditioned immune-deficient (NRGS) mice (2x105cells/mouse). Preliminary results from bone marrow aspirates of transplanted mice at weeks 4 post-transplant showed abundant human leukemia cells in mice receiving control cells while the mice receiving LAMP5-knockdown cells showed near-absence of human leukemia cells. Collectively, these results demonstrate that LAMP5, a novel target of MLL-fusion proteins is required for the propagation of leukemia. In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it localizes to the ER-Golgi intermediate compartment (ERGIC). In leukemia cells, using immunofluorescent confocal microscopy we detected LAMP5 in the perinuclear zones where it co-localized with ERGIC-53, a marker of the ERGIC compartment. While little is known about the functions of LAMP5 in normal pDC, studies suggest that it functions as a co-chaperone with UNC93B1, a known Toll-like Receptor (TLR) chaperone, to shuttle the TLRs to their respective locations in the plasma membrane or endosomes. In ongoing experiments, we are determining the functions of LAMP5 in leukemia, including its association with UNC93B1 and with the TLR-NFKB signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we postulate that LAMP5 could potentially serve as a therapeutic target with a wide therapeutic-window to treat MLL-leukemias. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document