Modification of Gene Expression in Mesenchymal Stromal Cells of the Leukemia Patients during Chemotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5065-5065
Author(s):  
Tamara Sorokina ◽  
Irina Shipounova ◽  
Alexey Bigildeev ◽  
Nina I. Drize ◽  
Larisa A. Kuzmina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Leukemia ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Leukemic Cell ◽  
Expression Level ◽  
Leukemic Cells ◽  
Disease Manifestation ◽  
Relative Expression Level

Abstract Background In patients with acute leukemia the stromal microenvironment is deeply modified. Disturbances in signaling pathways, genetic abnormalities and functional changes in mesenchymal cells of these patients have been previously described. Chemotherapy also affect stromal progenitor cells. A damaged microenvironment might impair hematopoiesis in acute leukemia patients. Aims To investigate the relative expression level in MMSCs and CFU-Fs, derived from the bone marrow (BM) of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients before and over the course of chemotherapy. Methods 54 newly diagnosed cases (33 AML, 21 ALL) were involved in the study after informed consent. BM was aspirated prior to any treatment (time-point 0) and at days 37, 100 and 180 since the beginning of treatment of acute leukemia. MMSCs were cultured in aMEM with 10% fetal calf serum, CFU-Fs, in aMEM with 20% fetal calf serum. The relative expression level (REL) of different genes was measured by TaqMan RQ-PCR. As a control MMSCs and CFU-Fs from 88 healthy donors were used. Results At the time of the disease manifestation the analysis of gene expression in MMSCs from acute leukemia patients revealed a significant increase in the REL of genes which regulate immune system responses and thereby can influence on the leukemic cell proliferation and migration (IL-6, IL-8, IL-1b and IL-1R1) (Pic.1). Also at the time of the diagnosis an increase in REL of genes, that are responsible for hematopoiesis regulation, was observed. For example, the REL of CSF1 that can influence on leukemic cells proliferation was increased at the disease manifestation and became normal during the treatment. The same dynamics was observed in the REL of JAG1 that has an antiapoptotic effect on leukemic cells. The REL of LIF had been also significantly increased at the disease manifestation, reflecting the efforts of MMSCs to inhibit leukemic proliferation. Chemotherapy affected REL of the studied genes differently. The treatment lead to the downregulation of IGF, TGFB1 and TGFB2 (Pic.2). As far asTGFB1 and 2 inhibit the differentiation of mesenchymal stem cells, and IGF is associated with myelodysplastic changes in elderly bone marrow, so their downregulation may refer to the effectiveness of therapy. The REL of genes regulating MMSC proliferation (PDGFRa and PDGFRb, FGF2, FGFR1 and 2) increased during chemotherapy. Exploring cell adhesion molecules, the decrease in the REL of their encoding genes was observed. As far as VCAM facilitate the leukemic cell extravasation and ICAM was shown to depress the Th17 cell differentiation, the down-regulation of their genes may reflect the microenvironment restoration. The influence of chemotherapy lead to decrease in REL of genes, associated with MMSCs differentiation (BGLAP and SOX9 (Pic.3)), reflecting the mechanism of the blocking of MMSCs migration and differentiation under the stress conditions. The alterations of bone marrow stroma were more pronounced in patients who didn't achieve remission. The REL of 9 genes was studied in CFU-F colonies. There were no differences in gene expression in CFU-Fs before the treatment, except for an increase in the REL of PPARg in acute leukemia CFU-Fs. During the treatment, a decrease in the REL of SPP1 and an increase in the REL of FGFR1 and 2 were observed. Conclusion Therefore, chemotherapy used does not impair the functional ability of MMSCs and CFU-Fs, but influence on their gene expression profile. The two types of precursors are affected differently, indicating their different differentiation level and functions. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Application of hyperthermia to the treatment of human acute leukemia: purging human leukemic progenitor cells by heat

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 802-804 ◽  
Author(s):  
Y Moriyama ◽  
M Narita ◽  
K Sato ◽  
M Urushiyama ◽  
S Koyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Progenitor Cells ◽  
Autologous Bone Marrow Transplantation ◽  
Leukemic Cell ◽  
Autologous Bone Marrow ◽  
Leukemic Cells ◽  
Neoplastic Disease ◽  
Myeloid Progenitor Cells

Abstract The application of hyperthermia to the treatment of neoplastic disease has focused on solid tumors. Since the hyperthermic sensitivity of human acute leukemia cells is not known, we have studied the in vitro response of human leukemic progenitor cells (L-CFU) to hyperthermia using a quantitative assay system for L-CFU. Human L-CFU were found to be more sensitive than committed normal myeloid progenitor cells to hyperthermic killing (41 to 42 degrees C). In addition, in the five acute myelogenous leukemic patients studied, it was shown that their leukemic progenitor cells--all types were studied according to the French-American-British diagnosis--were unable to form colonies when exposed to a temperature of 42 degrees C for 60 minutes, whereas the residual normal clones suppressed by the leukemic cell population were found to recover and to form more colonies in vitro as compared with untreated leukemic marrows. This strongly suggests that in vitro hyperthermia may selectively purge residual leukemic cells, especially L-CFU in stored remission bone marrow before autologous bone marrow transplantation.

Knock-in of a FLT3 Internal Tandem Duplication Mutation Cooperates with a NUP98-HOXD13 Fusion to Generate Acute Leukemia in a Mouse Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2961-2961
Author(s):  
Sarah M Greenblatt ◽  
Li Li ◽  
Christopher Slape ◽  
David Huso ◽  
Peter D. Aplan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Mouse Model ◽  
Acute Leukemia ◽  
Double Mutant ◽  
Tandem Duplication ◽  
Mutant Mice ◽  
Leukemic Cells ◽  
Internal Tandem Duplication ◽  
Cell Counts

Abstract Abstract 2961 Poster Board II-937 Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML), and provides a proliferative and survival advantage to leukemic cells. We have previously generated a knock-in mouse in which an 18-bp ITD mutation, isolated from a patient with AML, was inserted into the juxtamembrane domain of the murine FLT3 gene. Heterozygous FLT3WT/ITD mice develop a myeloproliferative disease, which progresses to mortality within 6 to 20 months. However, no sign of acute leukemia is observed over the lifetime of these mice, indicating that additional cooperating genetic events are required for leukemic progression. FLT3 activating mutations have been seen in MDS in about 5% of cases and an additional 5% of patients with MDS acquire FLT3 mutations as they progress to AML. One model of MDS has been developed by transgenic expression of the NUP98-HOXD13 (NHD13) fusion under the vav promoter. Mice expressing the NUP98-HOXD13 (NHD13) transgene develop a highly penetrant myelodysplastic syndrome (MDS) with about 50% eventually progressing to acute leukemia by 14 months. We wanted to generate a mouse model to see if FLT3/ITD mutations would cooperate with the NHD13 fusion to progress to overt leukemia. Double mutant mice (FLT3/ITD/NHD13) were generated by crossing heterozygous FLT3WT/ITD mice with mice expressing the NHD13 transgene. Strikingly, FLT3/ITD/NHD13 offspring (n=40) developed an acute leukemia with 100% penetrance and a median survival of 97 days. In contrast, NHD13 (n=20) and FLT3WT/ITD (n=20) littermates had median survivals of 385 and 410 days, respectively (Figure 1). Differential cell counts at the time of sacrifice showed elevated peripheral white blood cell counts in the double mutant mice (161.0±50.6 k/μl) compared to three-month-old wildtype (12.5 ±5.0 k/μl), NHD13(3.2 ±1.0 k/μl), or FLT3WT/ITD (7.9 ±3.5 k/μl) littermates. Organ sectioning and histological staining of double mutant mice showed leukemic infiltration of the spleen, liver, and brain. FLT3/ITD/NHD13 offspring developed a heterogeneous group of acute leukemias, with 46% developing ALL, 39% biphenotypic leukemia, and 15% AML as diagnosed by flow cytometry of the bone marrow and spleen. Transplantation experiments showed that the leukemia was able to engraft in lethally irradiated recipients, with disease occuring within 30 days post-transplantation. The disease was transferrable with as few as 1000 cells, and this ability was restricted to the B220+Mac-1−Gr-1- population. In order to identify the changes in gene expression responsible for leukemic transformation, RNA was isolated from the total bone marrow of young FLT3/ITD/NHD13, wildtype, NHD13, and FLT3WT/ITD littermates prior to the development of discernable disease and probed for over 43,000 coding and non-coding mouse sequences by Agilent 44K array. The rapid onset of acute leukemia in this model indicates the collaboration between hox gene dysregulation by a chromosomal translocation and altered signaling through the FLT3 receptor. In addition, since resistance to FLT3 inhibitors alone remains an important clinical issue, gene expression profiling of leukemic cells may help identify new molecular targets in collaborative signaling pathways. Disclosures: No relevant conflicts of interest to declare.

Differential Gene Expression in CLL Cells from Bone Marrow and Peripheral Blood Suggests a Role of Bone Marrow Stroma in Leukemic Cell Proliferation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 708-708
Author(s):  
Adrian Wiestner ◽  
Gerald E. Marti ◽  
Eric M. Billings ◽  
Hui Liu ◽  
Elinor Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Chemokine Receptors ◽  
Large Fraction ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Topoisomerase Ii Alpha ◽  
Extrinsic Factors

Abstract Chronic lymphocytic leukemia (CLL) has been described as an accumulative disease of mature lymphocytes. In the peripheral blood (PB) CLL cells are in Go/G1-phase, and gene expression is most consistent with resting B-cells. However, a recent study demonstrated that a surprisingly large fraction of CLL cells are constantly turned over and that proliferation contributes significantly to the expansion of the clone. The sites where proliferation occurs are not well defined but likely include the bone marrow (BM) and/or lymphoid organs. Likewise, the signals governing proliferation of the leukemic cells are ill defined but there appears to be a role for CLL extrinsic factors including stroma cell interactions or antigen stimulation. We hypothesized that gene expression in BM-CLL cells differs from that in PB-CLL cells. Here we report our analysis of 8 pairs of matched CLL samples derived from 7 individuals in whom we simultaneously obtained PB and BM. All patients were untreated, 3 female, 4 male; in 2 the CLL cells expressed unmutated IgVH genes, and 4 were ZAP70 positive. After informed consent, we obtained a dedicated research aspirate. CD19 selection resulted in >98% purity in all samples. PB-CLL cells express high levels of CXCR4, the receptor for SDF-1. As SDF-1 is expressed by BM stroma cells and CLL cells internalize CXCR4 after binding SDF-1 we measured CXCR4 expression by flow cytometry as an indicator of recent contact between leukemic cells and stroma. In PB, a mean of 63% (range 29–90%) of CLL cells expressed CXCR4 above isotype as compared to 30% (3–62%) of cells from the BM (p=0.007). Conversely, we followed CD69 expression as a marker of activation. In 5 of the 8 pairs CD69 was more highly expressed in BM than in PB derived CD19+ cells (p=0.005) indicating activation of the leukemic cells in the BM microenvironment. We performed gene expression analysis of total mRNA of all matched pairs on Affymetrix U133A 2.0 arrays according to standard protocols. We considered all genes with present calls in either PB-CLL or BM-CLL. The samples were normalized to correct for the individual to individual variance by first normalizing each individual’s PB and BM expression values by their PB value, and then averaging over the 8 individuals using GeneSpring software (Agilent). There were 543 genes with at least 1.5x higher expression in BM vs PB and 192 genes with at least 1.5x higher expression in PB versus BM at p<0.05. Genes more highly expressed in the BM derived CD19-selected cells included topoisomerase II alpha, several cyclins, including cyclin D1, signal transduction components such as PI-3 kinase and components of the Wnt pathway, transcription factors and enhancers such as c-Fos and Sox-4 and several chemokines and chemokine receptors. Of note, there was no difference in the expression of ZAP70, LPL, ADAM29, Bcl-2 and Mcl-1 between the two sites. Our findings are consistent with a model in which CLL cells migrate along an SDF-1 chemokine gradient to the BM where they are stimulated in contact with BM-stroma cells. The higher expression of cell cycle genes in the BM resident CLL cells supports recent findings of a sizeable proliferating fraction of CLL cells and suggests that at least part of this proliferating pool resides in the BM. Ongoing analysis is directed at identifying signaling pathways contributing such proliferation signals.

The Usefulness of CD71 Expression by Flow Cytometry in the Diagnosis of Acute Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2533-2533 ◽  
Author(s):  
Qian Liu ◽  
Mangju Wang ◽  
Yang Hu ◽  
Haizhou Xing ◽  
Xue Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Clonal Evolution ◽  
Expression Level ◽  
Leukemic Cells ◽  
Bone Marrow Blast ◽  
Blast Cells ◽  
The Mean ◽  
Acute Megakaryocytic Leukemia ◽  
Normal Controls

Abstract Abstract 2533 CD71 (transferring receptor 1) is an integral membrane glycoprotein that plays an important role in cellular uptake of iron. It is well known as a marker for cell proliferation and activation. Although all proliferating cells in hematopoietic system express CD71, however, CD71 has been considered as a useful erythroid-associated antigen. The expression proportion on nucleated red blood cells was significantly higher than other cells, approximately 80% of all CD71 positive cells were of CD71 positive erythroid cells in normal bone marrow. CD71 was usually considered as the representative marker for differentiating erythroblasts and diagnosing acute erythroid leukemia (AEL) by flow cytometry. At the ISAC 2000 Congress, most experts agreed that at least one or more B, T, myeloid, erythroid and megakaryocytic reagents should be included in the essential panel. The reagents recommended for erythroid cells included CD36, CD71 and glycophorin A (GlyA). However, there was no agreement on how to choose and group these antibodies. In the practical analysis of immune phenotypes of leukemic cells we noted that no CD71 expression was detected on blasts of some cases of AEL with typical morphological and cytochemical findings, but other types of acute myeloblastic leukemia (AML) cells may express CD71. Thus, we speculated that CD71 expression may associate with the abnormal antigen expression resulting from hematopoietic disorders. In this study, we evaluated CD71 expression on different acute leukemia cells in association with a variety of other antibodies. In this study we aimed to define CD71 as a flow cytometric marker for the diagnosis of acute leukemia. Bone marrow samples were collected from 82 newly diagnosed acute leukemia patients as well as 13 normal controls. The diagnosis were made according to the WHO 2008 diagnostic criteria. All 6 cases of AEL were erythroid/myeloid subtype (acute erythroid/myeloid leukemia, M6a). The samples were then analyzed using a four-color flow cytometer with antibody panels against a variety of lymphoid, myelomonocytic, erythroid and megakaryocytic antigens. The antibodies included anti-CD3, CD7, CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD45, CD56, CD61, CD64, CD71, CD117, GlyA, HLA-DR, IgG, IgM, MPO. Subpopulations of bone marrow cells were gated based on CD45 intensities and side scatter (SSC) value to further analyze the expression of antigens in different cell populations. Positive CD71 expression were identified on bone marrow blast cells of 41 (50%) acute leukemia patients and 9 (69.23%) normal controls. The mean expression level on normal controls was 35.99±19.06%. The mean CD71 expression level on blasts of AML with blasts differentiation at early stage of myelopoiesis (including FAB-M0/M1/M2/M4) was significantly higher than AML with partial differentiation of leukemic cells (FAB-M3/M5) and acuteB lymphoblastic leukemia (B-ALL) (p<0.05), with the mean expression level of 38.78±26.65%, 13.25±8.75% and 10.12±11.65%, respectively, and the latter two lower than normal controls (p<0.05). The percentage of CD71 expression level on blasts of acute megakaryocytic leukemia (FAB-M7) was 80.16±8.23%, significantly higher than normal controls, partial differentiation of leukemic cells (FAB-M3/M5), and B-ALL (p<0.05). The percentage of CD71 expression level on blasts of mixed lineage leukemia was 49.66±22.69%, significantly higher than B-ALL (p<0.05). Positive CD71 expression was found on bone marrow blast cells of 4 (66.67%) AEL cases, with the mean level percentage of 25.68±11.63% that was significantly lower than acute megakaryocytic leukemia (FAB-M7) (p<0.05) and was indifferent from normal controls and other types of acute leukemia. Using CD71 expression levels, we identified different abnormal cell clones simultaneously existing within bone marrow of 2 patients of AML with maturation (FAB-M2) and AEL, implicating the clonal evolution process from normal blasts to leukemic cells. CD71 is an important marker for diagnosing acute leukemia, and is useful for distinguishing the differentiation stages of AML. However, CD71 may not be the specific diagnostic marker for AEL. CD71 is also valuable for the observation of clonal evolution process of acute leukemia, which may be informative to the etiology of leukemia. Disclosures: No relevant conflicts of interest to declare.

CDX2 regulates Human Leukemogenesis Via Modulation Of The Wnt-Signaling Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3746-3746
Author(s):  
Anna Paczulla ◽  
Martina Konantz ◽  
Sarah Grzywna ◽  
Lothar Kanz ◽  
Claudia Lengerke
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Hox Genes ◽  
Wnt Signaling Pathway ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Nsg Mice ◽  
Cdx2 Expression

Abstract Introduction The caudal-type homeobox (Cdx) gene family has been mainly studied during early development for its role in axial elongation and antero-posterior patterning. More recently, Cdx genes were shown to regulate embryonic hematopoiesis by interactions with the canonical Wnt pathway and Hox genes. The role of Cdx genes in adult hematopoiesis remains poorly understood. Adult hematopoietic stem and progenitor cells derived from healthy murine bone marrow (BM) express low levels of Cdx1 and Cdx4 but not Cdx2. However, the majority (>80%) of human acute myeloid (AML) and lymphoid leukemias (ALL) were shown to express the human homologue CDX2, and ectopic induction of Cdx2 expression was sufficient to robustly induce myeloid leukemia in murine bone marrow cells. On the molecular level, the leukemogenic activity of Cdx2 was associated with modulation of Hox and Klf4 gene expression (Faber et al, 2013). The current study further explores the role of CDX2 in leukemogenesis by analyzing the effects of CDX2 expression induction or repression on human healthy and malignant hematopoietic cells and its molecular effects on the Wnt signaling pathway known to regulate Cdx genes during embryonic development. Methods Human bone marrow or mobilized peripheral blood derived CD34+ cells as well as the human leukemic cell lines SKM-1, NOMO-1, EOL-1 and NALM16 were exposed to lentiviruses containing CDX2 overexpression, shRNAs against CDX2 or control constructs. Efficient modulation of CDX2 expression was verified on gene expression level by qRT-PCR and on protein level by immunoblot analysis. CDX2 modified and control cells were subjected to growth, colony forming (CFU), cell cycle, flow cytometry and qRT-PCR gene expression analysis assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rγnull (NSG) mice. To explore the effect of Dickkopf-1 (DKK1), recombinant human DKK1 protein or carrier was supplemented to the methylcellulose in CFU assays. Results shRNA-mediated knockdown of CDX2 in leukemic cell lines lead to reduced growth (SKM-1, NALM16) and CFU formation (SKM-1 cells). Consistently, CDX2 knockdown SKM-1 cells showed lower ability to repopulate NSG mice and, upon subcutaneous injection in the flank, gave rise to much smaller tumors when compared to control cells, supporting the notion that CDX2 plays roles in human leukemogenesis. In contrast to the data published in mice, healthy human CD34+ cells transduced to overexpress CDX2 were unable to induce leukemia upon transplantation in NSG mice within an observation period of 5 months. On the molecular level, CDX2 modified cells showed differential expression of Klf4 and Hox but also Wnt pathway associated genes. Notably, robust induction of the canonical Wnt-inhibitory molecule DKK1 was observed in both healthy CD34+ stem/progenitor and leukemic cells upon CDX2 induction, while CDX2 suppression showed opposite effects. Analysis of the DKK1 promotor region revealed an interspecies conserved putative binding site for CDX2 as well as multiple HOX gene binding sites, suggesting that CDX2 can modulate DKK1 expression directly but also via its downstream HOX genes. Importantly, CFU assays performed on CDX2-knockdown cells showed a rescue of colony formation upon stimulation with DKK1 protein as compared to treatment with carrier only, demonstrating that the observed molecular interaction is functionally relevant in human leukemic cells. In contrast, control leukemic cells treated with DKK1 showed reduced CFU formation, indicating that CDX2 might act through DKK1 activation to fine-tune Wnt signal activation to the dosage that best promotes leukemogenesis and leukemic cell growth and survival. Conclusion Taken together, our data indicate that CDX2 employs DKK1 activation to modulate the Wnt signaling pathway and thereby growth, clonogenic capacity as well as in vivo tumorigenicity of human leukemia cells. In contrast to murine cells, CDX2 activation requires cooperative molecular events in order to induce leukemia in human healthy stem and progenitor cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Application of hyperthermia to the treatment of human acute leukemia: purging human leukemic progenitor cells by heat

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 802-804
Author(s):  
Y Moriyama ◽  
M Narita ◽  
K Sato ◽  
M Urushiyama ◽  
S Koyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Progenitor Cells ◽  
Autologous Bone Marrow Transplantation ◽  
Leukemic Cell ◽  
Autologous Bone Marrow ◽  
Leukemic Cells ◽  
Neoplastic Disease ◽  
Myeloid Progenitor Cells

The application of hyperthermia to the treatment of neoplastic disease has focused on solid tumors. Since the hyperthermic sensitivity of human acute leukemia cells is not known, we have studied the in vitro response of human leukemic progenitor cells (L-CFU) to hyperthermia using a quantitative assay system for L-CFU. Human L-CFU were found to be more sensitive than committed normal myeloid progenitor cells to hyperthermic killing (41 to 42 degrees C). In addition, in the five acute myelogenous leukemic patients studied, it was shown that their leukemic progenitor cells--all types were studied according to the French-American-British diagnosis--were unable to form colonies when exposed to a temperature of 42 degrees C for 60 minutes, whereas the residual normal clones suppressed by the leukemic cell population were found to recover and to form more colonies in vitro as compared with untreated leukemic marrows. This strongly suggests that in vitro hyperthermia may selectively purge residual leukemic cells, especially L-CFU in stored remission bone marrow before autologous bone marrow transplantation.

SET (TAF-IB) Gene Is Highly Expressed in Acute Leukemia Patients without Associated with Methylation Status.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4308-4308
Author(s):  
Sema Sirma ◽  
Cumhur G. Ekmekçi ◽  
Ayten Kandilci ◽  
Gerard Grosveld ◽  
Ugur Ozbek
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Leukemia ◽  
Gene Silencing ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Prognostic Significance ◽  
Gene Expression Level ◽  
Expression Level ◽  
Metastasis Suppressor

Abstract SET gene, also known as TAF-I beta, was originally identified as a component of the SET-CAN fusion gene, which results from t(9;9) translocation, in a patient with acute undifferentiated leukemia (AUL). SET gene encodes a nuclear phosphoprotein that ubiquitously expressed. There is an accumulating data that suggest a role for SET in gene silencing either through prevention of histone acetylation as a subunit of inhibitor of acetyltransferases (INHAT) complex or through inhibition of DNA demethylation. SET also inhibits the activity of protein phosphatase 2A, which involves in regulation of cell proliferation and differentiation, and blocks DNase activity of the tumor metastasis suppressor NM23-H1. Taken together, available data suggest that SET might play a role in tumorogenesis via tumor suppressor gene silencing or inhibition of apoptosis. In this study, we investigated SET gene expression level in bone marrow samples of 77 patients with acute leukemia (57 acute lymphoblastic leukemia (ALL) and 26 acute myeloid leukemia (AML)) and 5 control bone marrow samples from healthy volunteers using quantitative real-time RT-PCR. The ALL patient ages ranged 10months – 17 years, with a median of 6 years and the AML patient ages ranged 1–72 years, with a median of 18 years. For determination of the prognostic significance of SET gene expression in ALL patients, the association between patient’s clinical characteristics and the SET gene expression level was assessed usind the Pearson’s chi-square test or Fisher’s Exact test. Overall survival (OS) in 48 patients and relapse-free survival (RFS) in 37 patients with ALL at 6 years (median follow-up 35 months, range 1–79 months) were analyzed according to Kaplan-Meier method. We also studied methylation of various genes (p15, p16, p73, SOCS1, RAR beta, E-Cadherin, GSTP1, DAP-Kinaz, ER and 5-HIC) using methylation spesific PCR and COBRA analysis in AML samples to examine whether high SET gene expression play a role in tumorogenesis via gene silencing. Here we demonstrate that 54.4% of ALL patients and 53.8% of AML patients show two fold and higher SET expression level compare to control samples (p=0.005 and p=0.016 respectively). There were no significant association between SET gene expression level and age, peripheral WBC count, sex, FAB group and immunophenotype (P=0.823, P=0.182, P=1.00, P=0.132, P=0.751) in ALL. The OS was not significantly different between high (83.10% ± 6.92%) and low SET expressed patients (51.14% ± 18.73%) (log-rank=3.36, P=0.067) and the probability of RFS was not significantly different between high (81.20% ± 7.60%) and low SET expressed patients (80.00% ± 17.89%) with ALL (log-rank=0.01, P= 0.92). There was no statisticall association between methylation index and SET gene expression level (P=0.618). Our data suggest that high level of SET expression may play an important role in leukemogenesis. Further analyses are required to determine prognostic significance of SET gene expression different types of leukemia.

scholarly journals The Persistence of Extramedullary Leukemic Infiltrates during Bone Marrow Remission of Acute Leukemia

Blood ◽  
1965 ◽  
Vol 26 (2) ◽  
pp. 133-141 ◽  
Author(s):  
BOYD A. NIES ◽  
GERALD P. BODEY ◽  
LOUIS B. THOMAS ◽  
GEORGE BRECHER ◽  
EMIL J. FREIREICH
Keyword(s):  
Central Nervous System ◽  
Bone Marrow ◽  
Nervous System ◽  
Acute Leukemia ◽  
Lymph Nodes ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Common Site ◽  
The Central Nervous System

Abstract Leukemic cell infiltrates were found at autopsy in the tissues of 10 of 15 patients with acute leukemia dying during "complete bone marrow remission." The kidney was the most common site of leukemic cell infiltrates followed by the liver, testes, bowel, lung, central nervous system, and lymph nodes. These findings indicate that leukemic cells are not completely eradicated by current chemotherapy even in patients in whom no leukemic cells can be identified in the bone marrow. The distribution of residual leukemic cells demonstrates that the central nervous system is not the only reservoir of leukemic cells in patients during bone marrow remission.

scholarly journals Inhibition of Acute Leukemia By a Small Molecule KYA1797K Which Destabilize RAS and β-Catenin

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5758-5758
Author(s):  
Seungmin Hahn ◽  
Mi Ra Lee ◽  
Jungwoo Han ◽  
Moon Kyu Kim ◽  
Seung-Hwan Oh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Small Molecule ◽  
Wnt Pathway ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Beta Catenin ◽  
Downstream Targets ◽  
Flt3 Mutation

Introduction: Signaling pathways in acute leukemia are aberrantly activated to cause leukemogenesis and relapse after treatment. Like in many other malignancies, upregulation of WNT/beta-catenin pathway and hyperactive RAS is known to be associated with treatment resistance in leukemia. However, there are little studies about RAS singaling pathyway and leukemia, and it is a field of study that needs to be revealed. KYA1797K is a recently developed small molecule, binds directly to RGS domain of axin and enhances the beta-catenin destruction complex which activates GSK3beta and results in degradation of beta-catenin and RAS. In the current study, we tried to find the role of RAS inhibition by KYA1797K in leukemic cell lines and in patient's BM samples. Moreover, other small molecule PCK412 (Midosaturin) was also used for comparison. Materials & Methods: Leukemic cells (MOLT-4, THP-1, MOLT-4, Jurka, KG-1, MV4-11, RS4-11) were cultured in RPMI1640 media under various concentration (0.1-10µM for KYA1797K, 0.5-500nM for PKC412) for 48h and with Erlotinib (1µM) for comparison. Cell proliferation assay on each leukemic cell was done and immunoblotting for β-catenin, GSK3β, Pan-RAS, N-RAS was checked. Downstream targets of Wnt pathway (c-Myc, CD44, LEF1, Met, TCF1/TCF7) were studied by immunoblotting. MOLT-4 was stimulated with Wnt3a (200ng/mL, 4h) and changes in Wnt pathway were observed. Bone marrow samples of AML and ALL patients were evaluated for β-catenin and RAS. KYA1797K (Nat Chem Biol 2016, 12:593) was kindly provided by Prof. Kang-Yell Choi. Results: Suppression of leukemic cells by KYA1797K was evident starting from the concentration of 5 microM. (fig.1) Beta-catenin was down regulated in all cell lines by KYA1797K. Pan-RAS decreased in MOLT-4 and THP-1. All the downstream targets evaluated were down regulated by KYA1797K in MOLT-4 culture, and was evident at the concentration of 5microM. (fig.2) Stimulation of Wnt pathway by Wnt3a was inhibited by KYA1797K. (fig.3) Bone marrow samples from 7 ALL patients showed various status of β-catenin and RAS expression. Two high-risk patients showed suppression of β-cateinin and N-RAS by KYA1797K. (fig.4) In MV4-11 (FLT3 mutant) and RS4-11 (FLT3 wild type), IC50 for PKC412 was higher in RS4-11 compared to MV4-11 while KYA1797K showed same IC50 in both cell lines. β-catenin and RAS downregulation was observed by KYA1797K. Effects of KYA1797K analyzed by qRT-PCR and immunoblot for FLT3, N-RAS, MEK, ERK, ETS2 showed that KYA1797K downregulates FLT3 in MV4-11 even though FLT3 is not the main target of action. It was less effective on RS4-11. (fig.5) In bone marrow samples of ALL patient with FLT3 mutation, KYA1797K 1µM showed effect in reducing leukemia cells. (fig.6) Conclusion: This preclinical study suggests that KYA1797K may be an option for patients with acute leukemia. KYA1797K effectively destabilized β-catenin and RAS in acute leukemia even under Wnt pathway activation. FLT3, N-RAS, MEK, ERK, ETS2 were down regulated by KYA1797K, hence KYA1797K has a potential application for acute leukemia with FLT3 mutation. Extended studies including further in vivo study are needed to build up a strategy in small molecule therapy to target RAS in acute leukemia. Although with limitation, we suggest RAS inhibitor as a potential drug for leukemia. Disclosures No relevant conflicts of interest to declare.

Changing Gene Expression Patterns during Early Treatment of B-Precursor Acute Lymphoblastic Leukemia May Be Predictive of Relapse.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 232-232 ◽  
Author(s):  
Valerie de Haas ◽  
Rob Dee ◽  
Goedele Cheroutre ◽  
Henk van den Berg ◽  
Huib Caron ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Expression Level ◽  
Leukemic Cells ◽  
Diagnostic Samples

Abstract Treatment of pediatric ALL is based on the concept of tailoring the intensity of treatment to a patients risk. Clinical studies have shown that it is possible to stratify patients according to the levels of minimal residual disease after induction therapy and early during further treatment, since it has been demonstrated that the MRD level is the best predictive level for disease outcome. More recently, it has been shown that gene expression profiles of leukemic cells at diagnosis might be correlated with outcome. In previous studies we reported that slow responding subclones represent the clones causative for a leukemic relapse in oligoclonal disease. Based on these results, we hypothesized that the gene expression profile of the slow responding subclones present after the first weeks of chemotherapy might be more predictive than the profiles of all leukemic cells at diagnosis. Twenty-four genes were selected; most signalling molecules, transcription factors and functions relevant for oncogenesis, drug resistance and metastasis. Selection of genes was based on the presently available data on prognostic cDNA microarry studies of cytogenetically defined subgroups of childhood ALL. In particular, we analyzed results of recently published studies that compared gene expression levels between diagnosis and relapse in B-precusor acute lymphoblastic leukemia. (Staal, 2003 and Beesley, 2005). Gene sequences were obtained from public databases. Genes were tested on different leukemic cell lines. For all cell lines differences in gene expression level were demonstrated. The same panel of genes was tested on diagnostic samples of 16 ALL patients, subsequently followed by investigation of paired diagnosis - day 15 - relapse samples of 3 relapsed ALL patients. Leukemic material was obtained from cryopreserved bone marrow samples. All leukemic cells were purified by MACS purification based on markers expressed on the tumour, i.e. CD34, CD19 and CD10. RNA extraction and cDNA synthesis was performed according to the TRIZOL protocol. Expression levels were determined in a SYBR Green based real-time PCR assay. We were able to show different gene expression profiles in the 16 tested diagnostic samples. For the paired samples from relapsed B-precursor ALL patients, the expression level of several genes at day 15 was different (ΔCT&gt;1) in regard to diagnosis. Moreover, the changed expression at day 15 was comparable to the expression level of this gene at relapse. We conclude that indeed we were able to demonstrate that some of the genes have a changing pattern of expression during early therapy (day15), a pattern which is comparable to the pattern of gene expression at relapse and which is different from the pattern at diagnosis. We also demonstrated that purification of the bone marrow samples is necessary to be certain that the gene expression shown is relevant for the leukemic cells and not contaminated by other cells, i.e. T-cells. Figure Figure

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