The Central Nervous System Microenvironment Influences the Leukemia Transcriptome and Enhances Leukemia Chemo-Resistance

Abstract Central nervous system (CNS) relapse is a significant cause of treatment failure among patients with acute lymphoblastic leukemia (ALL). Isolated CNS relapse occurs in ~3-8% of children with leukemia and accounts for 30-40% of initial relapses in some clinical trials. In addition, CNS-directed therapies are associated with seizures, secondary neoplasms, encephalopathy, and long-term endocrine, developmental, neurovascular, and cognitive deficits. While many studies have demonstrated that interactions between leukemia cells and components of the bone marrow microenvironment influence leukemia development, maintenance, and chemo-resistance, the role of the CNS microenvironment in leukemia is less well studied. To investigate the influence of the CNS niche in leukemia, we asked whether the CNS niche could impart unique and functionally important gene expression changes in leukemia cells. We transplanted NALM-6 human, pre-B leukemia cells into NSG mice without prior irradiation to avoid perturbing the bone marrow and CNS niches. After systemic leukemia development the mice were euthanized and leukemia cells were isolated from the bone marrow and CNS microenvironments. Gene expression profiling of ~700 cancer-associated genes (NanoString® Technology) identified 36 leukemia genes differentially expressed (30 up-regulated and 6 down-regulated; fold change ≥ 2 and FDR<0.05) in leukemia cells in the CNS microenvironment relative to the bone marrow. Furthermore, functional annotation revealed the up-regulated genes were involved in known leukemia and cancer pathways including MAPK, RAS, and apoptosis. We elected to further examine the gene PBX1 as it is a transcription factor with known roles in hematopoiesis, leukemia, and cancer biology. PBX1 contributes to Evi-1 mediated leukemia development in a murine model of leukemia and is a partner with TCF3 in the t(1;19) translocation that occurs in ~5% of pre-B ALL. Interestingly, this translocation is also associated with an increased risk for CNS relapse. We confirmed PBX1 mRNA up-regulation in leukemia cells either isolated from the CNS or in leukemia cells co-cultured with CNS-derived, murine choroid plexus cells by quantitative PCR. Supporting the generalizability of these results, additional human B-cell leukemia lines SEM and REH also exhibited PBX1 mRNA up-regulation in leukemia cells isolated from the murine CNS niche. Western blots of NALM-6 and SEM leukemia cells isolated from the mouse CNS as well as from leukemia cells co-cultured ex vivo with choroid plexus cells also showed PBX1 up-regulation at the protein level. Finally, culture of leukemia cells in either choroid plexus cell conditioned media or human cerebral spinal fluid (CSF) had minimal effects on PBX1 protein levels, suggesting that direct cell contact, rather than a soluble factor(s), contributes to PBX1 up-regulation in leukemia cells. Following confirmation of PBX1 up-regulation in leukemia cells within the CNS microenvironment, we next ectopically expressed PBX1, or GFP as a control, in leukemia cells to identify functional consequences of PBX1 expression in leukemia cells. Leukemia cells expressing PBX1 exhibited decreased sensitivity to cytarabine relative to control cells as measured by both proliferation and apoptosis assays. Furthermore, shRNA targeting PBX1, but not control shRNA, prevented PBX1 up-regulation in leukemia cells when co-cultured with choroid plexus cells and modestly, but significantly, attenuated the ability of choroid plexus cells to protect leukemia cells from cyatabine-induced apoptosis. Finally, although PBX1 had no effect on leukemia proliferation, it caused enhanced colony-forming ability in semi-solid media, consistent with increased leukemia self-renewal properties. Together these results suggest PBX1 up-regulation in leukemia cells may contribute to both leukemia self-renewal properties and chemo-resistance in the CNS niche. In summary, we believe this work illustrates the unique and functionally important effect that the CNS microenvironment has on leukemia cells. More comprehensive analyses of the leukemia transcriptome, genome, and proteome in the CNS niche will build upon this foundation and provide a more detailed understanding of the role of the CNS niche in leukemia biology. Finally, this approach may identify targetable CNS niche-mediated mechanisms of leukemia chemo-resistance and self-renewal. Disclosures No relevant conflicts of interest to declare.

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Pediatric Patients with Extramedullary Leukemia Involving the Central Nervous System Have a Superior Outcome.

Blood ◽

10.1182/blood.v112.11.920.920 ◽

2008 ◽

Vol 112 (11) ◽

pp. 920-920

Author(s):

Donna Johnston ◽

Todd Alonzo ◽

Robert Gerbing ◽

Beverly Lange ◽

William G. Woods

Keyword(s):

Central Nervous System ◽

Bone Marrow ◽

Overall Survival ◽

Nervous System ◽

Leukemia Cells ◽

Relapse Risk ◽

Cns Disease ◽

Cns Relapse ◽

Significant Difference ◽

The Central Nervous System

Abstract Purpose: Extramedullary leukemia (EML), formally known as chloroma, is discrete collections of leukemia cells outside of the bone marrow. EML is often seen within the central nervous system (CNS) and these are often treated in a similar fashion as leukemia cells within the cerebral spinal fluid (CSF). We previously demonstrated that the presence of leukemia cells within the CSF (CNS leukemia) does not affect overall survival. We sought to determine the outcome of patients with central nervous system EML and compare these patients with those with CNS disease and non CNS EML. Methods: Patients enrolled on Children’s Cancer Group protocols 2861, 2891, 2941 and 2961 being treated for de novo acute myeloid leukemia (AML) with intensive timing chemotherapy were classified for the presence of CNS disease as CNS1 (<5 wbc in the CSF without blasts), CNS2 (<5 wbc in the CSF with blasts), or CNS3 (≥ 5 wbc in the CSF with blasts), as well as EML in the CNS (eg orbit, brain, etc) or non-CNS EML (eg skin, lung, etc). These patient’s outcomes were then analyzed. Results: A total of 1459 patients treated with intensive timing chemotherapy were analyzed in this study. At diagnosis, 1113 (76%) were CNS1, 143 (13%) CNS2, 154 (11%) CNS3, 48 (3%) had CNS EML, 57 (4%) had a non-CNS EML, and only 6 patients (0.4%) with CNS EML had CNS3 status. Patients with CNS EML had a significantly higher overall survival from study entry compared to patients with non-CNS EML (83% vs 38%, p<0.001), and compared to CNS3 patients (83% vs 50%, p<0.001). The patients with CNS EML also had a significantly higher event free survival compared to patients with non-CNS EML (65% vs 34%, p<0.001), and compared to CNS3 patients (65% vs 34%, p<0.001). There was no significant difference in relapse risk, bone marrow relapse, isolated CNS relapse, or EML relapse comparing patients with CNS EML and non-CNS EML. CNS EML patients had a significantly lower relapse risk compared to CNS3 patients (29% vs 49%, p=0.025). There was not a significant difference in bone marrow relapse, isolated CNS relapse, or EML relapse comparing these 2 groups of patients. Conclusion: Patients with extramedullary leukemia involving the CNS had a significantly better survival than patients with non-CNS EML or patients with CNS leukemia at diagnosis. This should reassure clinicians caring for these often challenging patients.

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PS913 ROLE OF CHOROID PLEXUS STROMA IN TUMOUR CHEMORESISTANCE: A SANCTUARY FOR LEUKAEMIA CELLS IN THE CENTRAL NERVOUS SYSTEM

HemaSphere ◽

10.1097/01.hs9.0000561932.46135.a6 ◽

2019 ◽

Vol 3 (S1) ◽

pp. 412

Author(s):

L.M. Fernández-Sevilla ◽

J. Valencia ◽

M.A. Flores ◽

A. Fraile-Ramos ◽

E. Jiménez ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Choroid Plexus ◽

The Central Nervous System

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Compartmentalization of HIV-1 in the central nervous system: role of the choroid plexus

AIDS ◽

10.1097/01.aids.0000166090.31693.aa ◽

2005 ◽

Vol 19 (7) ◽

pp. 675-684 ◽

Cited By ~ 35

Author(s):

Evan J Burkala ◽

Jun He ◽

John T West ◽

Charles Wood ◽

Carol K Petito

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Choroid Plexus ◽

The Central Nervous System ◽

Hiv 1

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The Role of TWIST1 Gene Expression and Hypoxic Microenvironment in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.3839.3839 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3839-3839

Author(s):

Emilia Carolina Malafaia ◽

A. Mario Marcondes ◽

Ekapun Karoopongse ◽

Daniele Serehi ◽

Maria de Lourdes L. F. Chauffaille ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Twist1 Expression ◽

Disease Biology ◽

Acute Myeloid

Abstract TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors (QIN Q et al., 2012) and has been described as an emerging risk factor in hematological neoplasms (MERINDOL et al., 2014). . Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to disease biology and therapeutic targets for patients with AML. Objectives: 1) to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients. Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak¨ 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan¨ Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of β-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V-phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information . Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test. Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1) showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04) (fig 1). We found increased TWIST1 mRNA levels in bone marrow samples of 23 AML patients (3.88 ± 1.59) compared with 5 healthy controls (0.54 ±0.25) (p= 0.02) (fig 2). Patients in the highest tertile of TWIST1 expression did not show differences in percentage of blasts in bone marrow and complete remission after treatment compared with patients in low and middle tertile. Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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The Potential Role of the Six1/Eya1 Pathway in the Establishment of Leukemia Stem Cells in MLL-ENL – Induced Leukemia

Blood ◽

10.1182/blood.v118.21.1362.1362 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1362-1362 ◽

Cited By ~ 1

Author(s):

Sylvia Takacova ◽

Pavla Luzna ◽

Viktor Stranecky ◽

Vladimir Divoky

Keyword(s):

Bone Marrow ◽

Cellular Transformation ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Transcription Profile ◽

Self Renewal ◽

Kinetics Of

Abstract Abstract 1362 During the multistep pathogenesis of acute leukemia (AL), a pool of leukemia stem cells (LSCs) emerges that is capable of limitless self-renewal and ensuring disease maintenance. The molecular mechanism that controls the kinetics of cellular transformation and development of LSCs is largely unknown. Using our MLL-ENL-ERtm mouse model, we have previously shown (Takacova et al., Blood 2009, 114 (22): 947–947, ASH abstract) activation of the ATR/ATM-Chk1/Chk2-p53/p21 checkpoint leading to senescence at early stages of cellular transformation (myeloproliferation), thereby preventing AL development in vivo. Experimental ATM/ATR inhibition accelerated the transition to immature cell states, acquisition of LSC properties and AL development in these mice. The MLL-ENL-ERtm mouse model allows us to study the kinetics of MLL-ENL-ERtm LSC development. We raised the questions how the transformation process progresses from the pre-LSC to the LSC state, and how DNA damage response (DDR) - mediated senescence affects the transition in gene expression. Given that the threshold of DDR signaling events is rate-limiting, we determined the transcription profile of the pre- LSC–enriched cell states derived from bone marrow and spleen of the MLL-ENL-ERtm mice at the early disease stage, and we correlated this transcription profile with the level of DDR, proliferation rate and induction of senescence. Pair-wise comparisons revealed up-regulation of the Six1 transcription factor gene and its cofactor Eya1 in the MLL-ENL-ERtm pre-LSCs in association with aberrant proliferation in both tissues. The notable difference between the two tissues concerning the barrier induction was the higher threshold of DDR and senescence in the bone marrow due to cooperation with inflammatory cytokines that fine-tune the DDR level. Interestingly, the expression of Six1 and Eya1 genes was down-regulated in senescence exclusively in the bone marrow. Consistent with these in vivo data, we found Six1 expression decreased in response to inflammation/DDR-induced senescence in the MLL-ENL-ERtm bone marrow cells cultured in vitro and correlated with SA-beta-gal positivity and p16 up-regulation. Six1 mRNA level was decreased only transiently after ionizing radiation (4 Gy)-induced DDR in the same cell line. These data suggest that Six1 expression is down-regulated in response to high DDR and permanent cell-cycle arrest in the MLL-ENL-ERtm pre-LSCs. Furthermore, we identified the transcription profile of the LSC-enriched cell state after inhibition of DDR in caffeine-treated MLL-ENL-ERtm mice in vivo. Interestingly, the expression level of Six1 and Eya1 was significantly increased in the bone marrow and spleen of the MLL-ENL-ERtm AML mice compared to the early (preleukemia) stage. High expression of Six1 and Eya1 and higher cell number expressing these genes was further confirmed by immunohistochemical staining on tissue sections. The MLL-ENL-ERtm LSC-enriched spleen cells showed increased colony forming ability in vitro and leukemia-initiating potential in serial transplantation experiments compared to pre-LSCs. Moreover, we detected Six1 and Eya1 expression in the infiltrating leukemia cells in tissues of the caffeine-treated MLL-ENL-ERtm AML mice and in a subset of leukemia cells in transplanted mice. Based on these findings and correlations, we hypothesized that the Six1/Eya1 pathway might be involved in regulation of some of the aspects of LSC development as well as invasion and maintenance of leukemia in our MLL-ENL-ERtm mice. Notably, our data indicate that senescence represses a subset of the MLL-ENL-downstream transcription response and prevents full activation of self-renewal. Experiments leading to more detailed understanding of the role of the Six1/Eya1 pathway in the MLL-ENL-induced cellular transformation are ongoing. Disclosures: No relevant conflicts of interest to declare.

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Extramedullary Hematopoiesis in the Choroid Plexus of Five Dogs

Veterinary Pathology ◽

10.1177/030098589503200417 ◽

1995 ◽

Vol 32 (4) ◽

pp. 437-440 ◽

Cited By ~ 18

Author(s):

D. Bienzle ◽

J. M. Kwiecien ◽

J. M. Parent

Keyword(s):

Central Nervous System ◽

Bone Marrow ◽

Nervous System ◽

Choroid Plexus ◽

Fourth Ventricle ◽

Extramedullary Hematopoiesis ◽

Vascular Supply ◽

The Other ◽

The Central Nervous System ◽

Refractory Seizures

Five dogs euthanatized because of refractory seizures were found to have hematopoietic elements in the interstitium of the choroid plexus at the level of the fourth ventricle. None of the dogs had significant hematologic or cerebrospinal fluid abnormalities. The extramedullary hematopoiesis was confined to the central nervous system and consisted of megakaryocytes, immature granulocytes, and rubricytes in two dogs and of one predominant cell population in each of the other three dogs. These findings are unique, and factors possibly contributing to the formation of a hematopoietic inductive microenvironment in the choroid plexus are cytokine-neurokine homologies, locally altered vascular supply, and aberrant functioning of bone marrow-derived central nervous system macrophages.

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Id1 Regulates the Choice of Hematopoietic Stem Cells to Self-Renew or Commit to Differentiation.

Blood ◽

10.1182/blood.v106.11.270.270 ◽

2005 ◽

Vol 106 (11) ◽

pp. 270-270 ◽

Cited By ~ 1

Author(s):

Vladimir Jankovic ◽

Alessia Ciarrocchi ◽

Piernicola Boccuni ◽

Robert Benezra ◽

Stephen D. Nimer

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Cell Surface ◽

Self Renewal ◽

Myeloid Progenitor ◽

E Protein

Abstract Id proteins belong to the basic helix-loop-helix family of transcription factors and act as dominant negative forms of E-protein transcriptional activators. Id mediated E protein silencing has an essential role in restricting differentiation and maintaining self-renewal in embryonic stem cells. However, the role of Id1 in adult stem cells including HSCs has not been described thus far. Having detected relatively high levels of Id1 mRNA in murine adult HSCs (compared to the committed myeloid progenitor cells) we examined the in vivo HSC function in Id1 deficient mice. We observed a >2 fold reduction in HSC frequency in the bone marrow in 8-10w old Id1−/− mice compared to Id1+/+ animals, detected by both lin-c-kit+Sca-1+ (LKS) cell surface marker profile and Hoechst 33342 dye efflux - “side population” phenotype, as well as a ~25% decrease in total bone marrow cellularity. Although Id1 deficient HSCs show robust long-term competitive repopulating capacity in primary transplant recipients, they have markedly impaired hematopoietic function upon secondary transplantation. Id1 null HSCs show a higher rate of S-phase entry in vivo as measured by 3 day BrdU incorporation (ko: 79.0±3.9% vs. wt: 49.7±7.4) and faster initial doubling times in response to cytokine stimulation in vitro during the first 2 days of culture. This failure to maintain normal HSC numbers and the diminished repopulating capacity, in the presence of enhanced cell cycling, suggests a defect in the regulation of self-renewal in Id1 deficient HSCs. Considering the general function of Id1 as an inhibitor of differentiation, the observed effect of Id1 loss could be explained by the excessive recruitment of LKS cells into the actively proliferating differentiated progenitor pool, at the expense of their self-renewal capacity. Consistent with this, sorted Id1−/− HSCs show accelerated expression of cell surface lineage markers in vitro and an increased ratio of CFU-S8 /CFU-S12 in the in vivo spleen colony forming assay. Global gene expression profiling of Id1+/+ vs. Id1−/− hematopoietic cells (using Affymetrix MOE430 Plus chips) revealed insignificant transcriptional deregulation in the committed myeloid progenitor subsets (CMP, GMP, MEP) in the absence of Id1. Meanwhile, Id1−/− HSCs showed a marked change in gene expression pattern (more than 1500 genes with a ≥2 fold difference in expression levels). Differentially regulated transcripts in Id1+/+ vs. Id1−/− HSCs significantly overlap (~30%) with the observed changes in gene expression that accompany the transition of HSCs to the common myeloid progenitor phenotype. Specifically, genes such as c/EBPα and GATA1 are significantly upregulated in Id1 null immunophenotypic HSCs, consistent with an earlier than normal commitment to myelo-erythroid differentiation. In contrast, several known transcriptional regulators of HSC self-renewal (Bmi1, Gfi1, HoxB4) show no significant change in expression pattern. These data clearly indicate the unique role of Id1 in regulating HSC self-renewal by restricting the rate of HSC commitment to the myeloid progenitor cell fate.

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Important Role of Routine Cerebrospinal Fluid Examination in Diagnosing Central Nervous System Relapse during Maintenance Therapy in Pediatric Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v106.11.870.870 ◽

2005 ◽

Vol 106 (11) ◽

pp. 870-870

Author(s):

Lani Lieberman ◽

Ronald Grant ◽

Johann K. Hitzler ◽

Adam Gassas

Keyword(s):

Central Nervous System ◽

Bone Marrow ◽

Cerebrospinal Fluid ◽

Nervous System ◽

Maintenance Therapy ◽

Lymphoblastic Leukemia ◽

Oncology Group ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Cns Relapse ◽

Asymptomatic Children

Abstract Despite the success of central nervous system (CNS) preventive therapy in reducing the incidence of CNS recurrence in pediatric acute lymphoblastic leukemia (ALL) on therapy, CNS relapse continues to be a significant cause of treatment failure and is observed in 5–10% of patients. While most pediatric ALL treatment protocols mandate periodic lumbar puncture (LP) surveillance for the length of therapy, some centers have stopped collecting routine CSF samples unless there are suggestive signs or symptoms of CNS involvement. Our objective herein was to assess the value of routine cerebrospinal fluid (CSF) obtained while performing routine LPs for administration of intrathecal chemotherapy in diagnosing CNS relapse in children with ALL in the maintenance phase. Patients and Methods: All children (0–18 years) with ALL, diagnosed and treated at the Hospital for Sick Children, Toronto, Canada between 1994–2004 were subjected to a retrospective analysis. Original reports for CNS relapse during maintenance therapy were examined to determine whether CNS relapse was diagnosed based on routine CSF sample obtained while administering intrathecal chemotherpy or a CSF sample obtained based on signs and symptoms or after a diagnosis of a bone marrow relapse. Results: Four hundred and ninety four children were diagnosed and treated in our institution during the study period. Children were treated based on the children oncology group (COG), pediatric oncology group (POG) and local protocols where applicable. Thirty-one children (6.3%) suffered CNS relapse while on maintenance therapy. Twenty-one had an isolated CNS relapse and ten had a combined bone marrow and CNS relapse. Seventy-six percent (16/21) isolated CNS relapses were diagnosed base on routine CSF samples obtained from asymptomatic children while administering intratheacal chemotherapy. Conversely, all patients with combined bone marrow and CNS relapse presented with symptoms and signs that warranted CSF examination. Conclusion: Routine CSF examinations are important in detecting CNS relapse in children with ALL during maintenance therapy. Furthermore, routine CSF sampling may detect isolated CNS relapse in asymptomatic children with ALL prior to extension into combined relapse where prognosis is less favorable.

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