Functional and Genomic Characterization of the Interaction between Acute Lymphoblastic Leukemia Cells and the Microenvironment Identifies Pathways for Therapeutic Intervention

Abstract Introduction Residence and interaction with a specialized bone marrow microenvironment is important for normal hematopoietic stem cells and for initiation and progression of myeloid malignancies, but the role of the microenvironment in propagation and therapeutic response of acute lymphoblastic leukemia (ALL) is not well known. Prior work has identified the efficacy of inhibiting FAK signaling, which is deregulated by IKZF1 alterations resulting in induction of THY1-Integrin alpha 5 adhesion in Ph-positive (Ph+) ALL. Here, we hypothesized that this mechanism may be more broadly important in ALL. We applied a systematic integrated genomic/imaging/functional approach to define the nature of interaction and identify changes in leukemic cells upon interaction that may be targetable. Materials and methods Time-lapse confocal imaging was performed to examine how leukemia cells migrate and adhere to mesenchymal stem cells (MSCs). NALM6 (DUX4/ERG), MHH-CALL2 (hypodiploid), 697 (TCF3-PBX1), Reh (ETV6-RUNX1) and SUP-B15 (Ph+) cell lines were cultured with immortalized human bone marrow MSCs transduced with telomerase reverse transcriptase (hTERT) (Mihara, Br J Haematol. 2003;120:846). For RNA-sequencing, non-adherent cell line cells were collected after two days of coculture with hTERT while adherent cells were trypsinized and collected. Both samples were sorted for CD19 positive population. Fresh primary ALL samples were cultured on bone marrow MSCs derived from patients with no hematological disease and collected with the same procedure for RT-PCR. Multicolor immunofluorescence imaging was utilized to observe expression of multiple molecules involved in adherence. Results Time-lapse imaging showed that leukemia cells have a dynamic interaction with MSC monolayers, with temporary adherence, accompanied by dynamic change in their shape. NALM6 cells adherent to MSCs reduced cell cycling, with an increase in the ratio of G0/G1 cells (26.7% to 48.0%) and decrease in S phase (60.7 to 41.8%). Analysis of gene expression showed 138 upregulated genes (log2FC >2 and FDR <0.05) in adherent cells which were common in all five cell lines, with striking upregulation leading to gene expression associated with gene ontology of extracellular matrix organization and collagen fibril organization. Representative genes were validated in adherent NALM6 cells by immunoblotting (FN1, TIMP1, and LGALS1). Pathway analysis showed that transforming growth factor beta 1 (TGFB1) was the top ranked upstream regulator (p-value of overlap 5.26E-35) in that 44 of 65 genes had measurement direction consistent with activation of TGFB1 signaling. In addition, other upstream regulators that may be involved were beta-estradiol, fibroblast growth factor 2, and tumor necrosis factor. Adhesion of leukemia cells to stroma may induce integrin expression and downstream signaling. Our transcriptomic analysis showed that integrins (A5, B1, A3 and B5) and caveolin 1 (CAV1), a main component of the caveolae plasma membranes, are highly transcribed in adherent leukemia cells. As hTERT cells showed unexpectedly low expression of THY1, a common MSC marker, we utilized primary bone marrow MSC for subsequent analysis. Immunoblotting assay showed enhanced expression of CAV1 in all cell lines adhered to MSCs. Multicolor immunofluorescence imaging demonstrated CAV1 and ITGB1 expression on leukemia cells that localized adjacent to stromal cells, which confirms that these molecules were upregulated upon adhesion to MSCs. Moreover, primary ALL cells showed remarkable upregulation of CAV1, ITGB1, ITGA3, and ITGB5 when the cells were adherent to MSCs. Conclusions Our results demonstrate that ALL cells dynamically interact with microenvironment cells, inducing changes in cell morphology, cell cycling, and adhesion, which may facilitate altered responsiveness to therapy. Transcriptional results suggest that TGFβ signaling is an upstream regulator after cell adhesion to MSCs. While integrins and CAV1 mediate the signaling, these pathway and molecules will be candidates for exploring inhibitors of signaling, which may affect their interaction and make them novel therapeutic targets. Figure. Figure. Disclosures Mullighan: Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

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Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽

10.1182/blood.v124.21.3569.3569 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3569-3569

Author(s):

Ye Shen ◽

Chun Shik Park ◽

Koramit Suppipat ◽

Takeshi Yamada ◽

Toni-Ann Mistretta ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Tumor Suppressor ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Brdu Incorporation ◽

Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

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Amino Acid Stress Response Genes Promote L-Asparaginase Resistance in Pediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-152940 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3304-3304

Author(s):

Daniel Ferguson ◽

J. Robert McCorkle ◽

Qian Dong ◽

Erik Bonten ◽

Wenjian Yang ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Acid Stress ◽

Leukemia Cells ◽

P Value ◽

Newly Diagnosed ◽

Advisory Committees ◽

Primary Leukemia

Abstract Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcome. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood, beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP resistant and sensitive ALL cell lines (n = 7) as well as primary leukemia samples from newly diagnosed patients. We identified 2219 response genes (absolute log 2FC > 1.5, FDR p-value <0.05) with ~16.5% being reproduced in more than one cell line. Defining target genes of the amino acid stress response related transcription factor ATF4 in ALL cell lines using ChIP-seq revealed 25% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor. A total of 17,117 significantly differentially bound ATF4 sites were identified (FDR p-value <0.01) and 97.8% of these sites displayed an increase in ATF4 binding following LASP treatment. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells from 212 newly diagnosed children enrolled on St. Jude Total Therapy 16. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlights that many response genes may not differ in their basal expression in drug resistant leukemia cells. Disclosures Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Mullighan: Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Pfizer: Research Funding. Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee. Evans: Princess Máxima Center for Pediatric Oncology, Scientific Advisory Board, Chair: Membership on an entity's Board of Directors or advisory committees; BioSkryb, Inc.: Membership on an entity's Board of Directors or advisory committees; St. Jude Children's Research Hospital, Emeritus Member (began Jan 2021): Ended employment in the past 24 months.

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Xenografts of Pediatric Acute Lymphoblastic Leukemia Retain the Gene Expression Pattern From the Diagnostic Material They Originated From Over Serial Passages.

Blood ◽

10.1182/blood.v114.22.1629.1629 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1629-1629

Author(s):

Manon Queudeville ◽

Elena Vendramini ◽

Marco Giordan ◽

Sarah M. Eckhoff ◽

Giuseppe Basso ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Leukemia Cells ◽

Diagnostic Samples ◽

Xenotransplantation Model

Abstract Abstract 1629 Poster Board I-655 Primary childhood acute lymphoblastic leukemia (ALL) samples are very difficult to culture in vitro and the currently available cell lines only poorly reflect the heterogeneous nature of the primary disease. Many groups therefore use mouse xenotransplantation models not only for in vivo testing but also as a means to amplify the number of leukemia cells to be used for various analysis. It remains unclear as to what extent the xenografted samples recapitulate their respective primary leukemia. It has been suggested for example that transplantation may result in the selection of a specific clone present only to a small amount in the primary diagnostic sample. We used a NOD/SCID xenotransplantation model and injected leukemia cells isolated from fresh primary diagnostic material of 4 pediatric ALL patients [2 pre-B-ALL, 1 pro-B-ALL (MLL/AF4}, 1 cortical T-ALL] intravenously into the lateral tail vein of unconditioned mice. As soon as the mice presented clinical signs of leukemia, leukemia cells were isolated from bone marrow and spleen. Isolated leukemia cells were retransplanted into secondary and tertiary recipients. RNA was isolated from diagnostic material and serial xenograft passages and gene expression profiles were obtained using a human whole genome array (Affymetrix U133 2.0). Simultaneously, immunophenotypic analysis via multicolor surface and cytoplasmatic staining by flow cytometry was performed for the diagnostic samples and respective serial xenograft passages. In an unsupervised clustering analysis the diagnostic sample of each patient clustered together with the 3 derived xenograft samples, although the 3 xenograft samples clustered stronger to each other than to their respective diagnostic sample. Comparison of the 4 diagnostic samples vs. all xenograft samples resulted in a gene list of 270 genes upregulated at diagnosis and 8 genes upregulated in the xenograft passages (Wilcoxon, p< .05). The high number of genes upregulated at diagnosis is most likely due to contamination of primary patient samples with normal peripheral blood and/or bone marrow cells as 15% of genes are attributed to myeloid cells, 7% to erythroid cells, 7% to lymphoid cells, 32% to bone marrow in general as well as to innate immunity, chemokines, immunoglobulins. The remaining genes can not be attributed to a specific hematopoetic cell lineage and are not known to be related to leukemia or cancer in general. Accordingly, there are no statistically significant differences between the primary, secondary and tertiary xenograft passages. The immunophenotype analysis are also in accordance with these findings, as the diagnostic blast population retains its immunophenotypic appearance during serial transplantation, whereas the contaminating CD45-positive non- leukemic cells disappear after the first xenograft passage. The few genes upregulated in xenograft samples compared to diagnosis are mainly involved in cell cycle regulation, protein translation and apoptosis resistance. Some of the identified genes have already been described in connection with cancer subtypes, their upregulation therefore being indicative of a high proliferative state in general and could argue towards a more aggressive potential of the engrafted leukemia cells but alternatively could also simply be due to the fact that the xenograft samples are pure leukemic blasts and are not contaminated with up to 15% of non-cycling healthy bone marrow cells as in the diagnostic samples. We conclude that the gene expression profiles generated from xenografted leukemias are very similar to those of their respective primary leukemia and moreover remain stable over serial retransplantation passages as we observed no statistically significant differences between the primary, secondary and tertiary xenografts. The differentially expressed genes between diagnosis and primary xenotransplant are most likely to be due to contaminating healthy cells in the diagnostic samples. Hence, the NOD/SCID-xenotransplantation model recapitulates the primary human leukemia in the mouse and is therefore an appropriate tool for in vivo and ex vivo studies of pediatric acute leukemia. Disclosures No relevant conflicts of interest to declare.

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Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells

PLoS ONE ◽

10.1371/journal.pone.0256708 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0256708

Author(s):

Christopher von Beek ◽

Linnéa Alriksson ◽

Josefine Palle ◽

Ann-Marie Gustafson ◽

Mirjana Grujic ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Acute Leukemia ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Tumor Formation ◽

Leukemia Cells ◽

Human Leukemia ◽

Bone Marrow Biopsies

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

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Identification of Gene Expression Profiles in Acute Lymphoblastic Leukemia Cells That Discriminate Intracellular Thioguanine Nucleotide Accumulation in ALL Cells after In Vivo Treatment with Mercaptopurine.

Blood ◽

10.1182/blood.v104.11.453.453 ◽

2004 ◽

Vol 104 (11) ◽

pp. 453-453

Author(s):

Gianluigi Zaza ◽

Meyling Cheok ◽

Wenjian Yang ◽

Pei Deqing ◽

Cheng Cheng ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Post Treatment ◽

Leukemia Cells ◽

True Positive

Abstract Thioguanine nucleotides (TGN) are considered the principal active metabolites exerting the antileukemic effects of mercaptopurine (MP). Numerous clinical studies have reported substantial inter-patient variability in intracellular TGN concentrations during continuation therapy of acute lymphoblastic leukemia (ALL). To identify genes whose expression is related to the intracellular accumulation of TGN in leukemia cells after in vivo treatment with MP alone (MP) or in combination with MTX (MP+MTX), we used oligonucleotide microarrays (Affymetrixâ HG-U95Av2) to analyze the expression of approximately 9,670 genes in bone marrow leukemic blasts obtained at diagnosis from 82 children with ALL. TGN levels were determined in bone marrow aspirates of these patients 20 hours after mercaptopurine infusion (1 g/m2 I.V). Because, as previously reported, patients treated with MP alone achieved higher levels of intracellular TGN compared to those treated with the combination, we used Spearman’s rank correlation to identify genes associated with TGN levels separately for the 33 patients treated with MP alone and the 49 with the combination (MP: median TGN: 2.46 pmol/5x106 cells, range: 0.01–19.98; and MTX+MP: median TGN: 0.55 pmol/5x106 cells, range: 0.005–3.31). Hierarchical clustering using these selected probe sets clearly separated the 33 patients treated with MP alone into two major groups according to TGN concentration (< 2.46 and > 2.46 pmol/5x106 cells; n=60 genes) and two major branches were also found for patients treated with the combination (< 0.55 and > 0.55 pmol/5x106 cells; n=75 genes). Interestingly, there was no overlap between the two sets of genes, indicating that different genes influence the accumulation of TGN when this drug is given alone or in combination with MTX. The association between gene expression profiles and TGN levels determined by leave-one-out cross-validation using support vector machine (SVM) based on Spearman correlation, was rho=0.60 (p<0.001) for MP alone and rho=0.65 (p<0.001) for MTX+MP, with false discovery rate (FDR) computed using Storey’s q-value (MP: 50% true positive, MTX+MP: 70% true positive respectively). Genes highly associated with the post-treatment TGN level in ALL patients treated with MP alone encode transporters, enzymes involved in the MP metabolic pathway and cell proliferation. Genes associated with post-treatment levels of TGN after combined therapy have been implicated in protein and ATP biosynthesis. Together, these in vivo data provide new insights into the basis of inter-patient differences in TGN accumulation in ALL cells, revealing significant differences between treatment with MP alone or in combination with MTX.

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Heme Oxygenase-1 in Leukemia Cells Promotes Cancer Associated Fibroblasts Mediating Progression of Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-130440 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3958-3958

Author(s):

Chengyun Pan ◽

Dan Ma ◽

Qin Fang ◽

Ping Liu ◽

Jishi Wang

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Bone Marrow Microenvironment ◽

Leukemia Cells ◽

Heme Oxygenase 1 ◽

Cancer Associated Fibroblasts ◽

Primary Cells ◽

Key Factor

Backgroud and objective: Recurrence and resistance is still the biggest challenges for Acute lymphoblastic leukemia (ALL). In recent years, a new concept has been proposed that the interaction between bone marrow microenvironment and leukemia cells could reduce the sensitivity of leukemia cells to chemotherapy. As an important matric element of the bone marrow microenvironment, Cancerassociated fibroblasts (CAFs) can mediate changes in bone marrow microenvironment to promote tumor proliferation and infiltration, but its role in ALL has not been described. Heme Oxygenase-1 (HO-1) is a rate-limiting enzyme in the process of heme catabolism, which is highly expressed in leukemia cells and can promote chemo-resistance by regulating the preservation of hematopoietic stem progenitor cells in the bone marrow microenvironment according to the recent research. In this article, we explored that HO-1 may be a key factor for promoting cancer associated fibroblasts to mediated chemo-resistance of ALL in the bone marrow microenvironment. M ethods : For clinical sample analysis, Bone marrow nucleated cells of 16 ALL patients with complete remission (ALL-CR), 12 ALL with relapse/refractory (ALL-R/R) and 10 normal controls were btained from bone marrow puncture. The expression of HO-1 and CAFs markers which includes α-SMA, FAP and FSP-1 was examined by quantitative reverse transcription-PCR (RT-PCR) and Western blot. In vitro cell experiments, CAFs was obtained from transfusion of bone marrow-derived mesenchymal stem cells (BM-MSCs) with recombinant human TGF-β1 (rhTGF-β1) stimulating. We used ALL primary cells and ALL cell lines (Nalm-6/Super-B15) to culture alone or co-culture with CAFs to detect proliferation, apoptosis and cell cycle of leukemia cells. In addition, we transfected ALL cell lines by constructing siHO-1 lentiviral vector, and co-cultured with CAFs or cultured separately to detect the above index changes in leukemia cells. R esults : The expressions of α-SMA, FAP, FSP-1 and HO-1 were significantly higher in ALL-R/R patients than in ALL-CR group and normal control group (P<0.05), which suggested that CAFs and HO-1 are closely related to recurrence and resistance of ALL. In vitro tests, after rhTGF-β1 stimulated BM-MSCs for 48h, the mRNA and protein expression levels of α-SMA, FAP and FSP-1 were significantly higher than those in BM-MSCs alone (P<0.05), This step was used to obtain CAFs. Then proceed to CAFs and Nalm-6/Super-B15 cell lines or ALL primary cells co-cultured tests. Results showed that CAFs can enhanced the leukemia cells proliferation and decreased apoptosis, and leukemia cells arrested in the G0/G1 phase was increased. Interestingly, this effect could be decreased by silencing HO-1 expression in Nalm-6/Super-B15 cell lines, which suggested that HO-1 may be a key factor mediating the interaction between leukemia cells and CAFs in bone marrow microenvironment. Conclusion:As an important component of the bone marrow microenvironment, CAFs are closely related to the recurrence and resistance of ALL. HO-1 may be a key component mediating the interaction between CAFs and leukemia cells of ALL. This results may provide a new entry point for deepening the mechanism of ALL progression and finding more effective targeted therapies. But further in vitro and in vivo experiments are still needed for verification. Disclosures No relevant conflicts of interest to declare.

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Development of a Unique In Vitro and In Vivo Model System of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph-ALL) with Emphasis on Cell to Cell Interaction.

Blood ◽

10.1182/blood.v108.11.1845.1845 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1845-1845 ◽

Cited By ~ 1

Author(s):

Arinobu Tojo ◽

Kiyoko Izawa ◽

Rieko Sekine ◽

Tokiko Nagamura-Inoue ◽

Seiichiro Kobayashi

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Suspension Culture ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Leukemia Cells ◽

L2 Cells

Abstract Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, readily acquires resistance to chemotherapeutic drugs including imatinib mesylate (IM), and shows a high relapse rate even after allogeneic stem cell transplantation. Nevertheless, primary blast cells are generally susceptible to apoptotic cell death in sort-term suspension culture after isolation from patients with Ph-ALL. We established two Ph-ALL cell lines and characterized their growth properties supported by adhesive interaction with a murine bone marrow stromal cell line, HESS-5. IMS-PhL1 (L1) cells mainly expressed p210-type BCR-ABL mRNA with wild type sequences in the ABL kinase domain and were weakly positive for p190-type mRNA. IMS-PhL2 (L2) cells exclusively expressed p190-type transcripts with Y253H mutation and showed much lower sensitivity to imatinib than L1 cells. The growth of L1 cells was slowly autonomous in suspension culture, but became more vigorous and their apoptosis was prevented by co-culture with HESS-5 cells. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5 cells and did not respond to soluble cytokines including SCF, IL3and IL7. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas. This migration was significantly inhibited by the pretreatment of those with a neutralizing antibody against α4-integrin. While non-adherent L1 cells were eradicated by 1 mM IM, a portion of adherent L1 cells could survive even at 10 mM IM. Similarly, adherent L2 cells considerably resisted prolonged exposure to 10 mM IM. Intravenous injection of both cell lines caused leukemia in NOD-SCID mice after distinct latent periods. Leukemia cells appeared in peripheral blood, bone marrow as well as spleen. Interestingly, expression of α5-integrin was significantly down-regulated in both leukemia cells collected from those tissues, but was restored after co-culture with HESS-5. The study of L1 and L2 cells in vitro and in vivo will not only contribute to further insights into microenvironmental regulation of clonal maintenance and progression of Ph-ALL but also provide a unique model for experimental therapeutics against Ph-ALL. Figure Figure

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

Blood ◽

10.1182/blood.v110.11.232.232 ◽

2007 ◽

Vol 110 (11) ◽

pp. 232-232 ◽

Cited By ~ 5

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>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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Acute Lymphoblastic Leukemia Cells Adapt to Post-Transplant Alloreactivity by Upregulating Sca-1.

Blood ◽

10.1182/blood.v112.11.1195.1195 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1195-1195

Author(s):

Jessica C Shand ◽

Andrew Campbell ◽

Johan Jansson ◽

Craig A. Mullen

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Mrna Expression ◽

Gene Expression Profile ◽

Expression Profile ◽

Lymphoblastic Leukemia ◽

Fold Increase ◽

Leukemia Cells ◽

Post Transplant ◽

Cells Cultured

Abstract Background: Relapse after allogeneic transplant represents the most common cause of treatment failure in patients with acute lymphoblastic leukemia, despite significant graftversus-leukemia effects. Objective: We wished to test the hypothesis that the gene expression profile of leukemia cells surviving in a setting of significant alloreactivity post-transplant differs from the gene expression profile of leukemia cells surviving in a setting of reduced alloreactivity. Methods: 10e4 GFP-labeled, C57/BL6-derived murine pre-B ALL cells carrying the human bcr/abl and INK4A/ARF mutations were incorporated into C3.SW hematopoetic cell grafts transplanted into normal C57/BL6 mice. These strains are MHC I- and II- matched, but differ at multiple minor histocompatibility antigens. The experimental group received cells from donors primed against recipients to produce a high graft-versus-host (GVH) disease environment. Control mice received naïve donor marrow. At 14–21 days post-transplant, leukemia cells were purified from marrow using immunomagnetic selection and, in a second experiment, flow sorting for enhanced purification. Affymetrix whole genome expression profiles were generated and analyzed using dCHIP software. Expression levels in a univariate analysis with ³ 2-fold changes over controls at p <0.0001 were considered significant. Expression of selected genes was further studied by flow cytometry and real-time PCR in leukemia cells cultured with or without interferon gamma, and in leukemia cells cocultured with B6-primed C3.SW bone marrow. Results: A small number of genes were consistently expressed at higher levels in ALL cells surviving in a high-GVH environment across both transplant experiments. These included interferon gamma (IFNg) dependent genes (interferon-inducible GTPase, +205.7 fold; interferon-induced transmembrane protein 3, +3.15 fold), hematopoetic growth factors (macrophage activation factor 2, +53.3 fold increase) and tumor suppressors (HRAS-like suppressor, +7.3-fold increase). Of particular physiologic interest was a 7.2 fold increase in Sca-1 (Ly6.A) expression. In the presence of IFNg in vitro, 91 ± 1.7 % of leukemia cells express Sca-1, compared with 52 ± 1.8 % of leukemia cells cultured without IFNg (p <0.0001, n=3). Comparably, there was a 1.3 fold increase in relative mRNA expression (p=0.0004, n=2) in these cells. Leukemia cultured with alloreactive (B6 primed) C3.SW bone marrow showed a 1.6 fold increase in Sca-1 surface expression (p =0.031) and a 2.0 fold increase in relative mRNA expression (p <0.0001) when compared with control leukemia cells. There was also a 1.2 fold increase in relative Sca-1 mRNA expression in leukemia cells cultured with alloreactive vs. naïve bone marrow (p = 0.0072). Conclusions: These data provide compelling evidence that leukemia cells change their gene expression profile in response to the immunologic pressure of the post-transplant environment. In vitro validation studies specifically demonstrate that Sca-1 is upregulated in the presence of IFNg and alloreactivity. Though the roles of Sca-1, as a hematopoetic lineage molecule and as a marker of activated lymphocytes, have traditionally been described separately, recent evidence suggests that Sca-1 is capable of coordinating multiple signals via lipid rafts. Upregulation of Sca-1 may therefore represent a powerful adaptive mechanism by which leukemia cells simultaneously promote their own hematopoetic colony formation and actively engage the host immune response. Further study of this novel mechanism has important implications for targeted leukemia therapy and immune modulation post-transplant.

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FGF2-Containing Exosomes Secreted from Bone Marrow Stromal Cells Protect Leukemia Cells from Tyrosine Kinase Inhibitors

Blood ◽

10.1182/blood.v126.23.2465.2465 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2465-2465

Author(s):

Jacqueline Martinez ◽

Nathalie Javidi-Sharifi ◽

Isabel English ◽

Shelton A. Viola ◽

Danielle Jorgens ◽

...

Keyword(s):

Bone Marrow ◽

Western Blot ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Leukemia Cells ◽

Advisory Committees ◽

Marrow Stroma ◽

Equity Ownership ◽

Fgfr Inhibition

Abstract Introduction. Mutational activation of kinases is a frequent event in leukemia, however resistance to kinase inhibitors remains a clinical dilemma. There is considerable evidence that proteins expressed by the bone marrow microenvironment protect leukemia cells from the effects of therapy. We previously reported that fibroblast growth factor 2 (FGF2) from bone marrow stroma protected chronic myeloid leukemia (CML) cells in a paracrine fashion. FGF2 expression was significantly increased in the marrow stroma of resistant CML patients without kinase domain mutations and resistance could be overcome with concomitant inhibition of FGFR (Traer et al. Blood 2014). Furthermore, resistant patients with increased marrow FGF2 expression had decreased FGF2 after FGFR inhibition, suggesting FGF2 also acts as an autocrine growth factor for stroma. Recently we have found a similar increase in marrow FGF2 in acute myeloid leukemia with FLT3 internal tandem duplication (FLT3+ AML), suggesting a more general mechanism of resistance (submitted). Since FGF2-mediated resistance appears to be conserved, we investigated FGF2 paracrine protection and autocrine stimulation in more detail. Results. FGF2 is expressed by stromal cells and plays an active role in hematopoiesis, however FGF2 does not have a signal peptide and thus its mechanism of secretion remains controversial. We used the related human stromal cell lines HS-5 and HS-27 to investigate FGF2 secretion (HS-5 strongly expresses FGF2 whereas HS-27 does not). FGF2 was found by Western blot to be enriched in the extracellular vesicle (ECV) pellet after centrifugation at 100,000g. These findings were confirmed by Luminex multiplex cytokine assay, where FGF2 was found to be uniquely enriched in ECVs. In order to further purify the ECV fraction, we performed a sucrose step-gradient fractionation and Western blot analysis. FGF2 was enriched in the exosome fraction, along with exosomal markers CD9 and tsg-101, whereas extracellular matrix proteins and apoptotic bodies localized to different fractions. Exosomes also conferred to K562 CML cell lines and MOLM14 FLT3+ AML cell lines treated with BCR-ABL and FLT3 inhibitors, respectively. To evaluate if FGF2 was contained within exosomes, we treated HS-5 exosomes with proteinase-K to digest proteins outside of the lipid membrane and found that FGF2 is present both inside and outside of exosomes. Exosomes were labeled with a fluorescent dye (DiI) and incubated with K562 and MOLM cells. Microscopy demonstrated uptake of exosomes into the leukemia cells. Protection by FGF2-containing exosomes could be partially abrogated by PD173074, a selective FGFR inhibitor, suggesting that protection by exosomes is not mediated entirely by FGF2, and other components of exosomes such as miRNAs and other proteins confer protection as well. To investigate FGF2 autocrine stimulation of marrow stroma, HS-5 and HS-27 cells were treated with PD173074. Growth of HS-5 cells was attenuated by inhibition of FGFR, whereas HS-27 cells were relatively unaffected. Treatment with PD173074 also caused distinctive changes in the morphology of HS-5. We then investigated the effects of FGFR inhibitor on FGF2 and exosome secretion. Although the intracellular FGF2 was unchanged by PD173074, the amount of secreted exosomes was decreased, as measured by FGF2, CD9 and tsg-101 by Western blot. This reduction in secreted exosomes was confirmed by NanoSight analysis, where increasing concentrations of PD173074 led to a dose-dependent decrease in secreted vesicles (Figure 1) indicating that exosome secretion is regulated by FGFR activation Conclusion. FGF2 signaling is a conserved mechanism of resistance to targeted therapy in CML, FLT3+ AML and other malignancies. FGFR inhibition by PD173074 leads to 1) reduced autocrine expansion of FGF2-expressing stroma, 2) decreased secretion of FGF2-containing exosomes, and 3) attenuation of the exosome-mediated protection of leukemia cells. Our findings suggest that exosomes are important purveyors of protective signaling to leukemic blasts in leukemia microenvironment, and that FGFR-inhibition may be a clinically relevant option to modulate the marrow stroma and overcome microenvironment-mediated resistance. Figure 1. Figure 1. Disclosures Druker: Henry Stewart Talks: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Sage Bionetworks: Research Funding; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Oncotide Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; ARIAD: Research Funding.

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