scholarly journals A Small Molecular Agonist of Histone Demethylase KDM3B Selectively Represses MLL-Rearranged Acute Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4682-4682 ◽  
Author(s):  
Xin Xu ◽  
Wilhelm G Dirks ◽  
Hans G. Drexler ◽  
Zhenbo Hu
Keyword(s):  
Dna Methylation ◽  
Acute Leukemia ◽  
Histone Acetylation ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Cell Lineage ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Background: Acute leukemia (AL) originates from both genetic and epigenetic changes that can be targeted to cure AL. Dysregulated DNA methylation has been shown to be associated with AL and demethylating agents 5-azacytidine and decitabine show favored improvement in secondary leukemia. Deficient histone acetylation has also been reported in AL and can be corrected to relieve leukemia. Histone methylation harbors more structural complexities compared to DNA methylation and histone acetylation and is broadly involved in AL. In particular, histone H3 lysine 9 (H3K9) methylation has been associated with AL. Di-methylation of H3K9 is reportedly involved in human hematopoietic stem cell lineage commitment. Moreover, tri-methylation of H3K9 predicts AML survival. H3K9 demethylation is catalyzed by exclusive KDM3 family members (KDM3A, KDM3B, and JMJD1C) that catalyze mono- and di-demethylation of H3K9, non-exclusive KDM4 family members (KDM4A, KDM4B, KDM4C, and KDM4D) that catalyze both H3K9 and H3K36 di- and tri-demethylation, KDM1A (LSD1) that catalyzes H3K4 and H3K9 mono- and di-demethylation, and PHF8 that catalyzes H3K9 mono- and di-demethylation and H4K20 demethylation. Among these, KDM3B, JMJD1C, KDM4C, LSD1, and PHF8 have been reported to be associated with AL in an enzymatic activity-dependent way. Furthermore, small molecular inhibitors of KDM4C and LSD1 have been developed for treatment of AML. H3K9 demethylase KDM3B is located at chromosome 5 band 31, a region frequently deleted or lost in acute myeloid leukemias (AML) and myelodysplasias (MDS). Different from other H3K9 demethylases that are usually responsible for leukemia maintenance, KDM3B harbors potential tumor-suppressive activities in acute myeloid leukemia and myelodysplastic syndromes. However, small molecular antagonists and agonists are lacking for KDM3B. Results: We aim to identify small molecular modulators of KDM3B. We focused on crystal structure of KDM3B Jumonji domain that catalyzes histone demethylation for virtual screening. From approximately 200,000 natural products and Chinese medicine components, we identified a potential KDM3B modulator, namely compound #7. Surface plasmon resonance technology showed that compound #7 binds to KDM3B with favorable affinity. In vitro and in vivo demethylation assay showed that compound #7 is able to increase H3K9 demethylating activity of KDM3B. We thus named compound #7 as KA-7 (KDM3B agonist #7). Interestingly, the identified KDM3B agonist KA-7 is able to selectively repress MLL-rearranged AL in cell proliferation and colony formation assays. Considering that KA-7 targets KDM3B that is located at chromosome 5q, a frequently deleted region in AML and MDS, we explored if KA-7 collaborates with Lenalidomide, an FDA approved drug for treating MDS with deletion at 5q where KDM3B is located. KA-7 was found to be able to synergistically increase the selective killing of AL cells by Lenalidomide. Conclusion: To sum up, physiologic H3K9 demethylase activity of KDM3B can be enhanced by a small molecular modulator KA-7 and causes selective killing against MLL-arranged AL cells. Disclosures. No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

Combined Single Cell Lineage and Transcriptome Sequencing Unveils Cell-Autonomous Regulators of Hematopoietic Stem Cell Fate

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 446-446
Author(s):  
Alejo E Rodriguez-Fraticelli ◽  
Caleb S Weinreb ◽  
Allon Moshe Klein ◽  
Shou-Wen Wang ◽  
Fernando D Camargo
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Large Fraction ◽  
Cell Lineage ◽  
Gene Signature ◽  
Hematopoietic Stem ◽  
Cell State

Blood regeneration upon transplantation relies on the activity of long-term repopulating hematopoietic stem cells (LT-HSCs). One of the major controversies in hematopoiesis relates to the apparently different properties that HSCs have in transplantation versus unperturbed settings. In unperturbed steady state hematopoiesis, the most potent HSCs appear to be mostly dormant, and only producing platelet-lineage cells. In turn, upon transplant, even a single transplanted HSC can actively divide and regenerate hundreds of millions of blood progenitors of all lineages. It would thus appear that HSCs have different fundamental properties in each study system. However, most transplantation studies have only tracked the lineage output of the transplanted HSC clones, and rarely the regeneration of the HSC compartment itself. In addition, clonal assays have not been performed at sufficient resolution to fully capture the diversity and clonal complexity of the regenerated HSC compartment. Here, we have used expressible barcodes, which can be sequenced in conventional single cell RNAseq assays, to simultaneously record the functional outcomes and transcriptional states of thousands of HSCs. Our analysis revealed multiple clonal HSC behaviors following transplantation that drastically differ in their differentiation activity, lineage-bias and self-renewal. Surprisingly, we witnessed a large fraction of clones that efficiently repopulate the HSC compartment but show limited contribution to differentiated progeny. Furthermore, these inactive clones have increased competitive multilineage serial repopulating capacity, implying that shortly after transplant a subset of clones reestablishes the native-like LT-HSC behaviors. Our results also argue that this clonal distribution of labor is controlled by cell autonomous, heritable properties (i.e. the epigenetic cell state). Then, using only our clonal readouts to segregate single HSC transcriptomes, we unveiled the transcriptional signatures that associated with unique HSC outcomes (platelet bias, clonal expansion, dormancy, etc.) and unraveled, for the first time, a gene signature for functional long-term serially repopulating clones. We interrogated the drivers of this cell state using an in vivo inducible CRISPR screening and identified 5 novel regulators that are required to regenerate the HSC compartment in a cell autonomous fashion. In conclusion, we demonstrate that functional LT-HSCs share more similar properties in native and transplantation hematopoiesis than previously expected. Consequently, we unveil a definition of the essential, common functional properties of HSCs and the molecular programs that control them. Figure 1 Disclosures No relevant conflicts of interest to declare.

Pak A Proteins Are Essential for Hematopoietic Stem/Progenitor Cell (HSC/P) Engraftment Through Regulation of Signaling Pathways Controlling HSC/P Proliferation, Survival, Migration, and the Actin Cytoskeleton

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 917-917
Author(s):  
Adrienne M. Dorrance ◽  
Rachelle Kosoff ◽  
Meaghan McGuinness ◽  
Chad Harris ◽  
Serena De Vita ◽  
...  
Keyword(s):  
Cell Migration ◽  
Conflicts Of Interest ◽  
Effector Proteins ◽  
Proliferating Cells ◽  
Hematopoietic Stem ◽  
Time Points ◽  
Empty Vector ◽  
Group A

Abstract Abstract 917 Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in HSC/P functions such as engraftment, retention, migration, adhesion, proliferation, and survival (Gu et al. Science, 2003). Our studies now focus on identifying potential Rac downstream effector proteins important for normal HSC/P function(s). The p21-activated kinases (Pak) are serine/threonine kinases that interact with and are major downstream targets of Rac and Cdc42. There are six human Paks (Pak1–6), which are grouped based on homology into Group A (Pak 1–3) and Group B (4–6) Paks. Paks regulate cytoskeletal organization including stress fiber dissolution, lamellipodia formation and focal adhesion disassembly and mediate activation of MAPK pathways. To identify the possible role(s) of Pak proteins in engraftment, freshly isolated LSK cells from WT (CD45.1+/CD45.2+) BM were transduced with retrovirus containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function or empty vector control (Mieg3); both constructs co-express GFP. 1.0×105 GFP+ LSK+ cells were then isolated and co-transplanted with 5.0×105 BoyJ (CD45.1+) whole bone marrow (WBM) into lethally irradiated C57Bl/6J (CD45.2+) recipients. Percent chimerism was measured at 3- to 24- weeks post BMT. PID transduced LSK+ cells were incapable of contributing to recipient hematopoietic reconstitution (Table 1). To explore the underlying mechanism of this engraftment failure we performed in vivo homing assays and found a 4- and 16- fold decrease, respectively, in BM homing of PID transduced LSK+ vs controls at 12 and 48 hours (p<0.05, for both time points). Altered cell migration of LSK+ cells was confirmed by live imaging microscopy which showed a 4-fold decrease in overall cell displacement in SDF-1-stimulated directed migration in the PID-expressing LSK+ compared to controls and was associated with a two-fold increase in random cell migration of PID-transduced LSK+ cells in transwell migration assays. PID-expressing LSK+ cells also demonstrated abnormal lamellipodia associated with significant increases in both cell surface area and cell perimeter. Because cytoskeletal changes may be linked to alterations in cell growth, we next examined the effect of Pak inhibition on cell survival and proliferation. PID-expressing LSK+ cells had decreased proliferation (17.7% vs 36.8% of cells in S-phase, p<0.05) and increased apoptosis (48.1% vs 16.7% AnnexinV+ cells, p<0.05) when compared to controls, respectively. These phenotypic changes were associated with decreased pERK and pAKT in PID-expressing LSK+. To confirm the importance of Pak activation of these proteins in HSC/P, we performed experiments to rescue the observed engraftment defect by co-transducing PID or Mieg3 with a constitutively active-ERK (ca-MEK1) or ca-AKT. We found ca-MEK1, but not ca-AKT, was able to increase proliferation in vitro (% proliferating cells for PID + empty vector = 6.1% and PID+ ca-MEK1 = 9.5%; p<0.05) and partially but only transiently rescue Pak-deficient HSC/P engraftment (% donor cells for LSK+ transduced with: PID + empty vector =1.5%, PID + ca-MEK1 =15.8%, and PID + ca-AKT =0.5% at 3-weeks post-BMT; p<0.05 for empty vector vs ca-MEK1). Finally, to determine which PakA pathway is critical in HSC engraftment we studied Pak genetic knock-out cells. We found that Pak2Δ/Δ -but not Pak1−/− -cells resulted in a profound HSC/P engraftment defect (% Pak2Δ/Δ vs Pak2flox/flox and Pak1−/− vs Pak1wt/wt: 1.0% vs 26.5% and 35.8% vs 37.4%; p<0.05 and p=ns, respectively at 3-weeks). Taken together, these data suggest that Pak A proteins regulate multiple HSC/P functions and link Rac GTPases to actin cytoskeletal rearrangements, directed cell migration, and proliferation/survival of HSC/P during engraftment.TABLE 1:Percentage of GFP+ cells in peripheral blood of recipient mice at indicated time points post BMT3-weeks6-weeks10-weeks14-weeks24-weeksWT-Mieg331.6% (±6.69)27.1% (±8.6)34.6% (±15.0)43.2% (±16.3)22.2% (±10.7)WT-PID0.22 (±0.19)0.07% (0.06)0%0%0%**Data represent mean ± s.d., n=10 recipients per group, p<0.05 for all time points, two independent experiments. Disclosures: No relevant conflicts of interest to declare.

TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4829-4829
Author(s):  
David C Dorn ◽  
Wei He ◽  
Joan Massague ◽  
Malcolm A.S. Moore
Keyword(s):  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Transforming Growth Factor Β ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2332-2332
Author(s):  
Vitalyi Senyuk ◽  
Yunyuan Zhang ◽  
Yang Liu ◽  
Ming Ming ◽  
Jianjun Chen ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Myeloid Differentiation ◽  
Dna Hypermethylation ◽  
Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

CXCR4 Invalidation Limits the MLL-ENL Induced Leucemogenesis in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4089-4089
Author(s):  
Yanyan Zhang ◽  
Hadjer Abdelouahab ◽  
Aline Betems ◽  
Monika Wittner ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Survival Time ◽  
Median Survival ◽  
Median Survival Time ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Abstract 4089 The receptor CXCR4 and its ligand SDF-1 play major physiological roles especially on hematopoietic stem cells homing and retention. Many studies have implicated CXCR4 in the invasion by tumor cells of organs that produce SDF-1. In acute myeloid leukemia, the physiological role of CXCR4 is not fully understood. We used retrovirus to express MLL-ENL oncogene in CXCR4+/+ and CXCR4−/− hematopoietic primitive cells (Lin- isolated from fetal liver) and showed that CXCR4 is dispensable for generation of immortalized colonies in vitro. To determine CXCR4 function in vivo, CXCR4+/+ and CXCR4−/− transformed cells were transplanted into lethally irradiated mice. Whatever their phenotype, the recipient developed a myelo-monocytique leukemia characterized by their expression of Gr-1 and Mac-1. As expected, all recipients of MLL-ENL transduced CXCR4+/+ cells were moribund within 35 to 80 days post transplant (median survival time: 50 days). Strikingly, recipients of MLL-ENL transduced CXCR4−/− cells showed significantly increased lifespan, with a median survival time of 90 days. The cellularity of the peripheral blood of recipients of MLL-ENL transduced cells displayed considerable increases over time although this increase was much lower in CXCR4−/− than in CXCR4+/+ chimera. Bone marrow of MLL-ENL transduced CXCR4−/− chimera had moderately decreased numbers of mononuclear cells. There were important numbers of leukemic CD45.2+/Gr1+/Mac1+/c-kit+ cells in spleen from MLL-ENL CXCR4+/+ chimera which suggested that CXCR4 is important for leukemic progenitors cells retention in the bone marrow and especially in the spleen. The homing capacity of transduced CXCR4+/+ cells is comparable to the CXCR4−/− cells. Finally, more DNA damages were found in the BM cells of MLL-ENL CXCR4−/− chimera. All these results were confirmed by treating of MLL-ENL CXCR4+/+ chimera with CXCR4 inhibitor (TN140). These results demonstrated that in absence of CXCR4, the cells transduced by oncogene MLL-ENL are capable of generating leukemia in the recipients. However, mice transplanted with MLL-ENL transduced CXCR4−/− FL cells developed acute myeloid leukemia with reduced aggressiveness and organ infiltration, which is associated with induced differentiation and DNA instability. These results indicated that the MLL-ENL progenitors are dependent on CXCR4 for their maintenance in the BM and spleen suggesting that CXCR4 inhibitors might have potential therapeutic applications. Disclosures: No relevant conflicts of interest to declare.

The Mechanisms of Synergistically Cytotoxicity Induced by Homoharringtonine and Aclarubicin in Acute Myeloid Leukemia Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4313-4313
Author(s):  
Lei Wang ◽  
Jie Jin
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Akt Pathway ◽  
Apparent Difference ◽  
Akt Inhibitor ◽  
Simultaneous Exposure ◽  
Acute Myeloid

Abstract Abstract 4313 Previous studies showed HAA regime [HHT (homoharringtonine), cytarabine and ACR (aclarubicin)] resulted in a high complete remission (CR) rate and a better overall survival (OS) rate in patients with primary acute myeloid leukemia. To confirm if a synergistically cytotoxicity was found in AML cells, we investigated the antitumor effect relationship of HHT and ACR against AML cells. Using in vitro system, we demonstrated that simultaneous exposure to HHT and ACR resulted in strong synergistic anti-proliferative effect and apoptosis inducing in AML cells. In vivo, combination of HHT and ACR may be result in a favorable survival in AML xenograft mice. The assay of microarray gene expressing profiling highlighted apparent difference in expression of PI3K gene and WNT3a gene between cells treated by HHT and cells exposure to ACR. Furthermore, decreased expression of PI3K110 and P-AKT protein were observed in AML cells treated with HHT for 3h while no significant change in the expression of two proteins was observed in 90nM of ACR-treated cells. Western Blot analysis also showed ACR could obviously inhibit WNT3a and β-catenin protein levels in AML cells after 3 hours exposure. Although HHT could not inhibit WNT3a protein, it also could apparently down-regulate expression of β-catenin in AML cells. Simultaneous decrease of PI3K signal and WNT3a signal was induced by the combination of HHT and ACR in AML cell lines and primary AML cells. To explore possible targets of synergistically cytotoxity induced by combined HHT/ACR, we silenced wnt3a expression by RNA interference. Then we found suppression of wnt3a expression could enhance the cytotoxity of HHT and AKT inhibitor. Moreover, combining ACR with AKT inhibitor resulted in a synergistically cytotoxic effect too. β-catenin is a shared molecular in both AKT pathway and WNT pathway. Up-regulating of β-catenin expression failed to reduce cell apoptosis induced by HHT plus ACR while partially decrease the growth inhibition rate caused by combining treatment. β-catenin is required for the self-renewal of AML-LSC. Our study also suggests that combining HHT and ACR may synergistically induce apoptosis in LSC-enriched cells. These results indicate that simultaneously inhibiting activity of PI3K/AKT pathway and WNT/β-catenin pathway is a possible mechanism of synergistically cytotoxity induced combinated HHT/ACR in AML cells. Disclosures: No relevant conflicts of interest to declare.

The BTK Inhibitor Ibrutinib Blocks SDF1/CXCR4 Mediated Migration of Acute Myeloid Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 915-915
Author(s):  
Stuart A Rushworth ◽  
Lyubov Zaitseva ◽  
Megan Y Murray ◽  
Matthew J Lawes ◽  
David J MacEwan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking ◽  
Acute Myeloid

Abstract Introduction Despite recent significant progress in the understanding of the biology of acute myeloid leukemia (AML) the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. Consequently, there is an urgent need to identify pharmacological strategies in AML, which are not only effective but can be tolerated by the older, less well patient. Recently our group and others have shown that there is high Bruton’s Tyrosine Kinase (BTK) phosphorylation and RNA expression in AML. Moreover, our recent study described for the first time that ibrutinib and BTK-targeted RNA interference reduced factor-induced proliferation of both AML cell lines and primary AML blasts, as well as reducing AML blast adhesion to bone marrow stromal cells. Inhibition of BTK has been shown to regulate chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma cell migration by inhibiting SDF1 (stromal derived factor 1) induced CXCR4 regulated cell trafficking. Here we report that in human AML ibrutinib in addition functions in a similar way to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. Methods To investigate the role of BTK in regulating AML migration we used both pharmacological inhibitor ibrutinib and genetic knockdown using a lentivirus mediated BTK targeted miRNA in primary AML blasts and AML cell lines. We examined migration of AML blasts and AML cells to SDF-1 using Transwell permeable plates with 8.0µM pores. Western blotting was used to examine the role of SDF-1 in regulating BTK, AKT and MAPK activation in primary AML blasts. Results We initially examined the expression of CXCR4 in human AML cell lines and found that 4/4 cell lines were positive for CXCR4 expression. Next we examined the effects of ibrutinib on the migration of the AML cell lines U937, MV4-11, HL60 and THP-1 in response to SDF1. We found that ibrutinib can inhibit the migration of all AML cell lines tested. We tested the in-vitro activity of ibrutinib on SDF-1 induced migration in a spectrum of primary AML blasts from a wide age spectrum of adult patients and across a range of WHO AML subclasses and found that ibrutinib significantly inhibits primary AML blast migration (n=12). Next we found that ibrutinib can inhibit SDF-1 induced BTK phosphorylation and downstream MAPK and AKT signalling in primary AML blast. Finally to eliminate the problems associated with off target ibrutinib activity we evaluated migration of AML cells lines using genetic inhibition of BTK. The introduction of BTK-specific miRNA dramatically inhibited the expression of BTK in THP-1 and HL60 and reduced SDF1 mediated migration confirming that BTK is involved in regulating AML migration in response to SDF1. Conclusions These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. Disclosures No relevant conflicts of interest to declare.

The Role of STAT5 in HOXA9-Associated Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3919-3919
Author(s):  
Peilin Ma ◽  
Yuqing Sun ◽  
Jingya Wang ◽  
Weihua Song ◽  
Tao Xu ◽  
...  
Keyword(s):  
Binding Sites ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Constitutive Activation ◽  
Oncogenic Mutation

Abstract Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that is essential for hematopoietic stem cell expansion and differentiation. Deregulation of HOXA9 is commonly observed in human acute myeloid leukemia (AML). About half of AML patients overexpress HOXA9 as a result of MLL rearrangements, NUP98 translocations, NPM1 mutations or CDX2/CDX4 overexpression. Despite its central importance in leukemia, the mechanism of transcriptional regulation by HOXA9 and its downstream effectors are poorly understood. HOXA9 physically interacts with MEIS1, a cofactor that greatly accelerates leukemia development in transplanted animals. Our group recently identified a number of transcription factors as HOXA9 potential collaborators by genomic profiling of HOXA9 binding sites and mass spectroscopy. One of these putative collaborators is signal transducer and activator of transcription 5 (STAT5), which coimmunoprecipitates with HOXA9. Furthermore STAT motifs extensively overlap with HOXA9 binding sites. STAT5 is important for survival, proliferation and differentiation of hematopoietic cells and constitutive activation of STAT5 has also been observed in human leukemias bearing oncogenic mutation of Jak2, Bcr-Abl, c-Kit and Flt3. FLT3 internal tandem duplication (FLT3-ITD) is observed in 25% of patients with MLL-partial tandem duplication (MLL-PTD) and is associated with HOXA9 upregulation and unfavorable prognosis. Therefore, we hypothesized that the interaction of HOXA9 and STAT5 may play a role in HOXA9-associated leukemogenesis. Treatment of human cell lines bearing MLL-AF9 and FLT3-ITD with specific FLT3 and STAT5 inhibitors showed that suppression of the constitutive activation of STAT5 significantly inhibits the hyper-proliferation of these cells. We then overexpressed FLT3-ITD or active mutation of STAT5 (STAT5 1*6) in mouse hematopoietic stem cells /progenitor cells (HSC/PCs) transduced with MLL-AF9 or HOXA9 and found that constitutively active STAT5 enhances cell proliferation in vitro. We next transduced HOXA9 into HSC/Pcs from wild type (WT) or FLT3-ITD transgenic mice and transplanted these cells into sublethally irradiated WT mice. All of these recipients developed myeloid leukemia, with recipients transplanted with FLT3-ITD (n=4) developing leukemia significantly earlier than WT controls (n=5, p<0.05), suggesting that FLT3-ITD mediated STAT5 activation enhanced HOXA9-induced leukemogenesis in vivo. To further assess the role of STAT5 in HOXA9-mediated transformation, we performed ChIP-Seq assay with HOXA9-transformed cells and identified nearly half of STAT5 binding sites (228 out of 596) colocalized with HOXA9. Most of these cobound sites are located in distal intergenic (61.0%) and intron (35.1%) regions. Five cobound regions (Il2rα, Fgf1, Pdlim5, Pim1, Fabp5) were selected and confirmed by ChIP-qPCR. To further characterize the interaction between HOXA9 and STAT5, GST pull-down assays were performed that showed that the c-terminal of HOXA9 is critical for interaction with STAT5. Overall, the findings suggest that STAT5 promotes HOXA9-induced transformation by functionally interacting with HOXA9 at HOXA9-regulated enhancers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2488-2488 ◽  
Author(s):  
José Gabriel Barcia Durán
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Fluorescent Microscopy ◽  
Hematopoietic Stem ◽  
Interleukin Receptors ◽  
Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

scholarly journals Variants ofDNMT3Acause transcript-specific DNA methylation patterns and affect hematopoiesis

2018 ◽  
Vol 1 (6) ◽  
pp. e201800153 ◽  
Author(s):  
Tanja Božić ◽  
Joana Frobel ◽  
Annamarija Raic ◽  
Fabio Ticconi ◽  
Chao-Chung Kuo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Acute Myeloid

De novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing ofDNMT3Ahas characteristic epigenetic and functional sequels. SpecificDNMT3Atranscripts were either down-regulated or overexpressed in human hematopoietic stem and progenitor cells, and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that, particularly, transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating thatDNMT3Avariants also affect malignancies. Our results demonstrate that specificDNMT3Avariants have a distinct epigenetic and functional impact. Particularly, DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in acute myeloid leukemia.

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