Genome-Wide shRNA Screen Implicates Mitogen Activated Protein Kinase (MAPK) Pathway In Glucocorticoid Resistance In Acute Lymphoblastic Leukemia

Abstract Resistance to glucocorticoids (GC) is a hallmark of relapsed acute lymphoblastic leukemia (ALL) and is a predictor of outcome at diagnosis. In spite of the importance of GC in the treatment of ALL and other hematological malignancies the molecular mechanisms that lead to effective eradication of leukemic cells is incompletely understood. To address this problem we have performed a functional screen for genes involved in prednisolone resistance in ALL cell line (Reh) and correlated these results with our previously published results using an integrated genomic analysis to discover genes (pathways) altered at relapse (Hogan et al 2011). Cells were infected with a pooled whole genome shRNA library that contained approximately 80,000 shRNAs targeting 18,000 genes. Deep sequencing was used to identify shRNAs enriched or depleted upon treatment with prednisolone. Three computational methods including; bioinformatics for next generation sequencing analysis (BiNGS), redundancy & fold change analysis (RFC) and strict standardized mean difference (SSMD) were applied to the sequencing data in efforts to obtain the most robust set of candidate genes for validation (Porter et. al, Leukemia 2012, Zhang XD, J Biomol Screen, 2007). Through our primary screen a total of 263 genes were identified to modulate prednisolone sensitivity in ALL. Upon knockdown, 142 genes increased the sensitivity of the cells to prednisolone and 121 genes increased resistance to prednisolone. Five genes overlapped with genes previously identified to be altered at relapse compared to matched diagnosis samples including SLC6A18, AARSD1, MIER3, CDC42BPB, and YAP1. We hypothesize that genes that are altered at relapse in ALL and identified through functional genomics screening to modulate chemosensitivity in vitro, are likely drivers of chemoresistance and eventual relapse. We also performed gene ontology (GO) analysis using DAVID Bioinformatics to identify pathways that may be responsible for altered resistance to prednisolone. This analysis strongly implicated the mitogen-activated kinase (MAPK) pathway. The MAPK was also identified as a pathway with increased activity at relapse through our integrative genomics analysis(Hogan et al 2011). One gene of particular interest was MAP2K4 which encodes for MEK4, an upstream kinase involved in JNK phosphorylation and c-Jun activation. Knockdown of MAP2K4 by shRNA in B-precursor ALL cell lines (Reh and RS4;11) results in statistically significant (p-value < 0.5) increased sensitivity to prednisolone induced apoptosis at a range of prednisolone concentrations but not to other chemotherapy tested (etoposide, doxorubicin, and 6-thioguanine).604. Molecular Pharmacology, Drug Resistance: Poster I Upon treatment with prednisolone MAP2K4 knockdown cells have increased levels of prednisolone responsive genes GILZ (1.4-2.4 fold in Reh, 2.1-3.8 fold in RS4;11) and TXNIP (1.8-5.7 fold in Reh, 2.5-2.6 in RS4;11). Increased sensitivity to prednisolone and increased levels of prednisolone responsive genes was associated with decreased levels of p-JNK that has been previously implicated in regulating glucocorticoid signaling through phosphorylation of the glucocorticoid receptor (GR) at S226 (Roatsky et. al, PNAS 1998, Itoh et. al, Mol. Endo. 2002).s Overall this data suggests that decreased levels of MAP2K4 results in increased sensitivity to GC by increasing GC signaling and implicates MEK4 as novel drug target in ALL. Disclosures: No relevant conflicts of interest to declare.

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Triptolide Induces Apoptosis In Acute Lymphoblastic Leukemia Cells by Inhibition of the Oncoprotein MDM2

Blood ◽

10.1182/blood.v116.21.4934.4934 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4934-4934

Author(s):

Mei Huang ◽

Lubing Gu ◽

Muxiang Zhou

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Biological Action ◽

Leukemic Cells ◽

Transcriptional Level ◽

Antitumor Effects ◽

Mdm2 Expression ◽

Pediatric All ◽

Chinese Plant

Abstract Abstract 4934 Triptolide, a nature product derived from the Chinese plant Tripterygium wilfordii, is reported to exhibit antitumor effects in a broad range of cancers. Recent studies indicate that the antitumor activity of triptolide is associated with its biological action to inhibit expression of many oncoproteins and anti-apoptotic or survival factors that were expressed in the cancer cells. Herein, we demonstrate that triptolide induces apoptosis in a subgroup of acute lymphoblastic leukemia (ALL) cells that overexpress MDM2 oncoprotein by inhibiting the MDM2 expression. In pediatric ALL, overexpression of MDM2 by leukemic cells is typically associated with a wild-type (wt) p53 phenotype and resistance to conventional chemotherapeutic drugs such as doxorubicin. In the present study, we evaluated the role of triptolide in regulating MDM2 and in inducing apoptosis, as compared to doxorubicin, using ALL lines and primary ALL samples. In contrast to doxorubicin, which induced p53 activation and a subsequent upregulation of MDM2, triptolide strongly induced persistent inhibition of MDM2 followed by a steady-state activation of p53, which resulted in potent apoptosis of the MDM2-overexpressing ALL cells tested, even if they were doxorubicin-resistant. We discovered that triptolide's inhibition of MDM2 in ALL cells occurred at the post-transcriptional level through inhibition of mRNA synthesis. Because p53 function is inhibited by MDM2 in chemoresistant/MDM2-overexpressing ALL cells, potent killing of these cells by triptolide suggests that this naturally-derived agent may be a novel therapeutic for refractory ALL. Disclosures: No relevant conflicts of interest to declare.

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Second Primary Malignancies In Adult Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.3871.3871 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3871-3871

Author(s):

Binay K. Shah ◽

Krishna B Ghimire

Keyword(s):

Acute Lymphoblastic Leukemia ◽

General Population ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Adult Patients ◽

P Value ◽

Second Primary ◽

Adult Age ◽

Absolute Excess Risk ◽

Second Primary Malignancies

Abstract Background Pediatric acute lymphoblastic leukemia (ALL) patients have higher rates of second primary malignancies. There is limited data on second primary malignancies (SPM) among adult patients with ALL. This study was conducted to evaluate SPM in adult ALL patients using US Surveillance, Epidemiology and End Results (SEER) cancer registry database. Methods We analyzed the SEER 13 Registries using multiple primary standardized incidence ratio (MP-SIR) session. We analyzed secondary cancer rates among adult ALL patients during the period 1992 - 2010. We used SEER*Stat software provided by national cancer institute for statistical analysis. Results There were 3,259 adult (age ≥20 years) ALL patients reported in SEER database during 1992-2010. Among them, 65 ALL patients developed 75 second primary malignancies. Fifty-nine ALL patients developed 1 SPM each, 3 ALL patients developed 2 SPM each, 2 ALL patients developed 3 SPM each and 1 ALL patient developed 4 SPM. All site cancers were significantly higher among adult ALL patients compared to general population with observed/expected ratio (O/E): 1.47, p value< 0.05, an absolute excess risk of 24.43 per 10,000 populations. Similarly, oral cavity cancer, respiratory system cancer and hematological SPM were significantly higher in ALL patients than expected in general population. (Table) Conclusions Adult patients with ALL have higher rates of second primary malignancies compared to general population. Disclosures: No relevant conflicts of interest to declare.

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JAK2 Aberrations in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.583.583 ◽

2016 ◽

Vol 128 (22) ◽

pp. 583-583

Author(s):

Elisabeth M.P. Steeghs ◽

Isabel S. Jerchel ◽

Willemieke de Goffau-Nobel ◽

Alex Q. Hoogkamer ◽

Judith M. Boer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Induced Resistance ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Leukemic Cell ◽

Leukemic Cells ◽

Cell Precursor

Abstract Background In high risk pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients, gain of function mutations and translocations affecting JAK2 have been described. These mutations and translocations result in aberrant kinase signaling and may therefore serve as an ideal target for precision medicines. Aim Evaluate the frequency and prognosis of JAK2 lesions among different subtypes of childhood BCP-ALL, and study the efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib. Methods This study comprised 77 BCR-ABL1-like cases and 76 B-other cases which were screened for JAK2 translocations using RT-PCR. Furthermore a representative pediatric cohort of 461 newly diagnosed BCP-ALL cases was screened for JAK2 mutations using targeted next-generation sequencing. Clinical analyses were performed in 341 BCP-ALL patients. Patient-derived-xenograft (PDX) cells were isolated from NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, which were injected with primary leukemic cells. Purity of PDX cells was enriched to over 90% and presence or absence of JAK2 lesions was validated. PDX and primary leukemic cells were exposed to a dilution series of momelotinib or ruxolitinib for four days. Where indicated, cells were pre-incubated with 25 ng/ml TSLP for 1 hour. In mono-culture assays, cytotoxicity was quantified using MTT and in co-culture assays flow cytometry was used. Leukemic cells were discriminated from mesenchymal stromal cells (MSCs) using CD19 and viability was assessed by Annexin V and Propidium Iodide. Western blotting was used to study protein expression levels. Results JAK2 translocations were detected in 6.5% of BCR-ABL1-like cases (3 PAX5-JAK2 cases, 1 TERF2-JAK2 case and 1 BCR-JAK2 case), but not in B-other cases. JAK2 mutations were identified in 3.5% of all BCP-ALL cases, which included JAK2 mutations in BCR-ABL1-like (7.6%), B-other (11.9%), and high hyperdiploid cases (1.6%), but not in MLL rearranged, BCR-ABL1-positive, ETV6-RUNX1-positive or TCF3-PBX1-positive cases. Cumulative incidence of relapse in patients harboring JAK2 lesions was as poor as in JAK2 wildtype BCR-ABL1-like and B-other patients. Efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib was examined in JAK2 lesion positive (primary and PDX) leukemic cells. Inhibitors were cytotoxic in both translocated and mutated cells, although efficacy in JAK2 mutated cells highly depended on CRLF2 activation by TSLP. CRLF2 activation resulted in downstream STAT5 activation and sensitization towards ruxolitinib compared to unstimulated cells (p < 0.05). Cells harboring JAK2 translocations signaled independently of CRLF2. Although momelotinib and ruxolitinib exposure blocked downstream STAT1/5 phosphorylation, both inhibitors also induced accumulation of phosphorylated JAK2Y1007. Consequently, release of the inhibitors resulted in a profound re-activation of JAK2 signaling, observed by upregulation of downstream STAT1/5 signaling. Furthermore, we observed microenvironment-induced resistance. Culturing leukemic cells in the presence of primary bone marrow MSCs induced resistance to ruxolitinib, compared to leukemic cells in single cultures (p < 0.05). A similar trend was observed for momelotinib. In addition, patients harboring JAK2 mutations displayed a heterogeneous leukemic cell population. Mouse xenograft models revealed different outgrowth patterns of leukemic cells, in which the JAK2 mutated clone persisted, decreased or even disappeared, resulting in outgrowth of JAK2 wildtype leukemic cells. Moreover, JAK2 mutations were not mutually exclusive for other pathway mutations (e.g. KRAS). Conclusion JAK2 translocations and mutations were detected in poor prognostic BCP-ALL cases. In ex vivo assays, the JAK1/2 inhibitors momelotinib and ruxolitinib were cytotoxic in JAK2 aberrant cells. Despite these promising findings, we identified certain limitations of these inhibitors. Inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon their release. Furthermore, our data suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and by microenvironment-induced resistance. Taken together, our data yield important directives for the clinical use of JAK inhibitors in pediatric BCP-ALL. Disclosures No relevant conflicts of interest to declare.

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A Key Role for Tumor Suppressor BTG1 In the Regulation of Metabolic Stress Responses: Implications for Asparaginase Resistance In Pediatric Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v116.21.1028.1028 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1028-1028

Author(s):

Laurens van der Meer ◽

Marloes Levers ◽

Liesbeth van Emst ◽

Joost C. van Galen ◽

Ad Geurts van Kessel ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Stress Responses ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Metabolic Stress ◽

Therapy Resistance ◽

Metabolic Enzyme ◽

Central Component ◽

Pediatric Acute Lymphoblastic Leukemia

Abstract Abstract 1028 Background: L-Asparaginase (ASNase) is a key component of multi-agent chemotherapy regimens used in the treatment of pediatric Acute Lymphoblastic Leukemia (ALL). This bacterially-derived enzyme lowers blood asparagine levels by catalyzing the hydrolysis of L-asparagine, leading to specific killing of leukemic blasts. However, relapses and associated therapy resistance occur in about 20% of the patients. Although the molecular mechanisms that contribute to ASNase resistance are not well understood, increased expression of asparagine synthethase (ASNS), the enzyme responsible for asparagine synthesis, has been linked to ASNase resistance both in ALL cell lines and in primary leukemias. We recently reported that micro-deletions affecting the B-cell translocation gene 1 (BTG1) occur in about 10% of pediatric pre-B ALL cases. Here we show that BTG1 loss contributes to ASNase resistance by up-regulation of ASNS. Results: Using RNA interference, we show that loss of BTG1 expression promotes cell growth and renders pre-B ALL cells completely refractory to ASNase induced cell death. Resistance to ASNase in the BTG1 knockdown cells is accompanied by increased expression of ASNS, while knockdown of this metabolic enzyme is sufficient to reverse therapy resistance, indicating that upregulation of ASNS is required for the observed resistance phenotype. We further show that BTG1 associates with and regulates the activity of the transcription factor ATF4, a key regulator of metabolic stress responses and a central component in the regulatory network that controls ASNS expression. Conclusion: Together, our experiments identify BTG1 as an important regulator of ASNS expression which acts by modulating ATF4 function. We expect that a detailed molecular understanding of how BTG1 loss contributes to ASNase resistance, will lead to the identification of pharmacological targets that can be used to improve treatment responses in therapy-resistant ALL. Disclosures: No relevant conflicts of interest to declare.

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Paired Immunophenotype Comparison Of Diagnosis and Relapse Samples In B-Lineage Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.4954.4954 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4954-4954

Author(s):

Marion Eveillard ◽

Richard Garand ◽

Nelly Robillard ◽

Soraya Wuilleme ◽

Caroline Thomas ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Leukemic Cells ◽

Aberrant Expression ◽

Blast Cells ◽

B Lineage

Abstract Although great progress has been made in the treatment of acute lymphoblastic leukemia (ALL), relapse remains a major issue in the follow-up of these patients. Recent data about the emergence of subclones during haematological malignancies suggest that relapses could result from resistant cells initially in minority or from cells driven to resistance by previous treatments. Among the tools allowing for the characterization of leukemic cells, flow cytometry (FCM) is an essential approach. Increasingly used to evaluate minimal residual disease (MRD) based on the immunophenotypic features of the blasts at diagnosis, it can also allow to identify immunophenotypic shifts related to clonal evolution. Such an approach would be best studied by comparing follow-up samples from the same patients. In order to be thorough, this would however require that conditions as similar as possible are applied to both types of cells. This work was designed 1) to compare the immunophenotypic features of B-ALL blast cells with those of normal hematogones, 2)to assess potential immunophenotypic shifts at relapse 3)to determine the stability of markers not classically used at diagnosis during follow-up and their potential utility for MRD. FCM was performed simultaneously on thawed paired samples from 15 patients (9 children aged between 1 and 12 years old and 6 adults aged between 23 and 71 years old) with B-lineage ALL. With a three-tubes 8 colours panel comprising a backbone of CD45, CD34, CD22 and CD10, the expression of 8 markers was examined and compared to that observed on normal hematogones contained in 29 bone marrow samples from healthy donors. These 8 markers were CD7, CD19, CD20, CD24, CD38, CD58, CD81, CD123. Moreover, an additional four colours panel was used to examine the more recently described antigens CD44, CD200, CD304 and Her2Neu. The presence of leukemia associated immunophenotypes (LAIP) was defined as a difference in mean fluorescence intensity (MFI) between hematogones and blasts of at least 2 standard deviations. At diagnosis, the expression of each marker was at variance from that on hematogones yielding LAIP in all patients, with at least 4 aberrant markers (up to 11). Antigens with the most abnormal expression were CD10 (100%), CD24 (93.3%), CD81 (80%), CD38 (60%) and CD58 (53.3%). Antigens with the least aberrant expression were CD19 (20%), CD22 (20%), CD123 (20%), CD34 and CD20 (46,7%). CD44 which is at a low level on hematogones, was present for 80% of the patients at diagnosis and overexpressed in ALL with MLL rearrangement (3/15 cases). CD200 was overexpressed in 73% of the patients while CD304 was present in only 40% of the patients. A single patient was positive for Her2Neu, which remained present at relapse. All patients retained at relapse the same global immunophenotype without any change in the EGIL classification (3 B-I, 8 B-II, 4 B-III) and the difference with hematogones remained. The expression of most markers was similar at diagnosis and relapse. There was no change at all for the expression of CD38 which therefore appears as the most interesting marker for follow-up and MRD in ALL. Only one patient each showed a change in the expression of CD44, CD58 or CD123. As a whole, stable markers were CD58, CD44, CD200, CD81 and CD24 in contrast with CD19, CD22, CD123, CD304, CD24 and CD20 which changed in 27 to 67% of the patients. Four patients displayed no immunophenotypic change at relapse while 3 showed a modification of a single marker. For 5 patients, with respectively 6 and 7 LAIP, two markers were modified at relapse. Three markers changed for the patient with Her2Neu expression. Finally, only two patients (13%) showed major changes possibly associated with the emergence of a new clone. This study confirms that B-ALL blast cells differ immunophenotypically from hematogones, although the latter have been reported to possibly be their normal counterpart. These data moreover comfort the interest of using LAIP in the detection of MRD in multiparameter FCM. Finally, since molecular targets of therapeutic monoclonal antibodies do not shift sensibly, their use can also be considered at relapse. Disclosures: No relevant conflicts of interest to declare.

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Chemotherapy resistance in acute lymphoblastic leukemia requires hERG1 channels and is overcome by hERG1 blockers

Blood ◽

10.1182/blood-2010-01-262691 ◽

2011 ◽

Vol 117 (3) ◽

pp. 902-914 ◽

Cited By ~ 87

Author(s):

Serena Pillozzi ◽

Marika Masselli ◽

Emanuele De Lorenzo ◽

Benedetta Accordi ◽

Emanuele Cilia ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Chemokine Receptor ◽

Molecular Mechanisms ◽

Lymphoblastic Leukemia ◽

Survival Rates ◽

Chemotherapy Resistance ◽

Leukemic Cells ◽

Channel Blockers ◽

Integrin Subunit ◽

Signaling Complex

Abstract Bone marrow mesenchymal cells (MSCs) can protect leukemic cells from chemotherapy, thus increasing their survival rate. We studied the potential molecular mechanisms underlying this effect in acute lymphoblastic leukemia (ALL) cells. Coculture of ALL cells with MSCs induced on the lymphoblast plasma membrane the expression of a signaling complex formed by hERG1 (human ether-à-go-go-related gene 1) channels, the β1-integrin subunit, and the chemokine receptor CXC chemokine receptor-4. The assembly of such a protein complex activated both the extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt prosurvival signaling pathways. At the same time, ALL cells became markedly resistant to chemotherapy-induced apoptosis. hERG1 channel function appeared to be important for both the initiation of prosurvival signals and the development of drug resistance, because specific channel blockers decreased the protective effect of MSCs. NOD/SCID mice engrafted with ALL cells and treated with channel blockers showed reduced leukemic infiltration and had higher survival rates. Moreover, hERG1 blockade enhanced the therapeutic effect produced by corticosteroids. Our findings provide a rationale for clinical testing of hERG1 blockers in the context of antileukemic therapy for patients with ALL.

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Clonal Origins of 'late' Relapses in ETV6-RUNX1 Acute Lymphoblastic Leukemia..

Blood ◽

10.1182/blood.v114.22.1583.1583 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1583-1583

Author(s):

Frederik W van Delft ◽

Sharon W Horsley ◽

Kristina Anderson ◽

Caroline M Bateman ◽

Susan Colman ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Copy Number ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Childhood All ◽

Remission Duration ◽

Clonal Origin

Abstract Abstract 1583 Poster Board I-609 Approximately a quarter of B cell precursor childhood acute lymphoblastic leukemia (ALL) is characterized by an ETV6-RUNX1 (TEL-AML1) fusion gene and has an overall good prognosis. The majority of these children will be treated on the standard risk arm of the United Kingdom ALL treatment protocols. Relapse usually occurs after cessation of treatment but remarkably can present many years later. The incidence of ETV6-RUNX1 at relapse has been reported to be less than or similar to de novo ALL. Molecular studies on neonatal bloodspots and on twins with concordant ALL have demonstrated the prenatal origin of major subtypes of childhood ALL, including most ETV6-RUNX1 fusion gene positive cases. In addition these investigations have suggested the existence of a preleukaemic stem cell requiring additional mutations or ‘hits’ in order to develop frank leukemia. To understand the genetic basis and clonal origin of late relapses we have compared the profiles of genome-wide copy number alterations (CNA) at relapse versus presentation in samples matched with remission DNA from 24 patients. The selected samples had tumor cell purity >75% before DNA extraction. DNA copy number alteration data was generated using the Affymetrix 500K SNP arrays. LOH analysis was performed using CNAG 3.0 and dCHIP 2008. Overall we identified 168 CNA at presentation and 252 at relapse (excluding deletions at IgH and TCR loci), equating to 6.96 and 10.3 CNA at presentation and relapse respectively. Although the number of CNA increased at relapse, no single gene or pathway was uniquely targeted in relapse. The most frequent alterations involved loss of 12p3.2 (ETV6), 9p21.3 (CDKN2A/B), 6q16.2-3 and gain of 21q22.1-22.12. A novel observation was gain of part or whole of chromosome 16 (2 patients at presentation, 5 at relapse) and deletion of the oncogene Plasmocytoma Variant Translocation 1 (PVT1) in 3 patients. Pathway analysis demonstrated frequent involvement at presentation and relapse of genes implicated in both B cell development (44 versus 46%) and cell cycle control (46 versus 71%). In order to study the clonal origin of relapse, we devised a classification describing the change in CNA between presentation and relapse in each individual patient. The clonal relationship between the presentation and relapse clone was established by the persistence of both the ETV6-RUNX1 fusion and at least 1 Ig and/or TCR rearrangement. We used a classification focussed on ‘driver’ CNA, defined as CNA that target genes functionally involved in leukemogenesis or CNA that are recurrently targeted as described in the literature. The four categories of relapse were type 1 (the dominant clone at presentation presented unchanged at relapse), type 2 (the relapse clone was derived from the major subclone at presentation with additional CNA), type 3 (the relapse clone was derived from a minor clone at presentation with gains and losses of CNA) and type 4 (the relapse clone is derived from an ancestral or preleukemic clone at initial presentation with all CNA gained). Twenty-one of the 24 patients were classifiable in this way (Figure 1). Although comparative relapse / presentation CNA profiles cannot identify precise clonal origins of relapse, the data indicate that irrespective of time to relapse (<2 to 9.9 years), the relapse clone appeared to be derived from either a major or minor clone at diagnosis with none (0/6) of the very late relapses (>5 years) derived from pre-leukemic cells lacking CNA. This data indicate diverse clonal origins of relapse and extended periods of dormancy, possibly via quiescence, for stem cells in ETV6-RUNX1+ ALL. Relapse type Remission duration (years) < 2 2 - 5 > 5 1 • • 2 • ••••••• •• 3 •• •• ••• 4 •• Figure 1. Each patient is represented by a black dot. Each patient is classified on the basis of the relapse type and remission duration. Disclosures No relevant conflicts of interest to declare.

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MicroRNAs Characterize Genetic Diversity and Drug Sensitivity in Pediatric Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v114.22.89.89 ◽

2009 ◽

Vol 114 (22) ◽

pp. 89-89 ◽

Cited By ~ 1

Author(s):

Diana Schotte ◽

Renee X de Menezes ◽

Farhad Akbari Moqadam ◽

Ellen Lange-Turenhout ◽

Caifu Chen ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Mirna Expression ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Mirna Expression Profile ◽

Leukemic Cells ◽

Expression Levels ◽

Genetic Subtypes

Abstract Abstract 89 MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate the activity of protein-coding genes, including those involved in cancer. The function of a miRNA depends on the cellular context and hence prominent miRNAs in lymphoma may not be important in acute lymphoblastic leukemia (ALL). To understand which miRNAs may be relevant in pediatric ALL, the expression levels of 397 miRNAs including seven newly cloned miRNAs were measured in seven genetic subtypes of ALL and normal hematopoietic cells. Except for BCR-ABL-positive and B-other ALL all major subtypes, i.e. T-ALL, MLL-rearranged, TEL-AML1-positive, E2A-PBX1-positive and hyperdiploid ALL, have unique miRNA signatures that differ from each other and from those in healthy hematopoietic cells. The expression of miR-383, miR-125b, miR-99a, miR-100 and let-7c was increased by a five to 1700-fold (P < 0.001) in TEL-AML1-positive cases. Hyperdiploid patients demonstrated a three to 24-fold upregulation (P < 0.001) of miR-222/222*, miR-223, miR-511 and miR-660, encoded on either chromosome X or 10 which is often duplicated in hyperdiploid cases. Some ALL subtypes shared similarities in their miRNA expression signature suggesting a common underlying biology e.g. both E2A-PBX1 and T-ALL cases demonstrated a downregulation of eight miRNAs (P ≤ 0.02) and within the TEL-AML1-positive subtype, two distinct groups were identified of which one showed an overlapping miRNA expression profile with hyperdiploid cases. Aberrant miRNA expression may result in dysregulated expression of their targeted proteins. Here we observed that the 70-fold downregulation of let-7b in MLL-rearranged ALL was associated with a 2-fold upregulation of oncoprotein c-Myc (P< 0.0001). Furthermore, a classifier built with a selection of 28 miRNAs predicted the MLL-rearranged, TEL-AML1-positive, E2A-PBX-positive and T-ALL subtypes with 100% sensitivity and specificity. Besides the genetic subtype, cellular drug resistance determines outcome of ALL. In vitro resistance of patients to vincristine, daunorubicin and L-asparginase was characterized by abnormal expression of 27 miRNAs (P < 0.05) whereas no discriminative miRNAs were found for resistance to prednisolone. Most striking was the 14- to 25-fold upregulation (P ≤ 0.002) of miR-125b, miR-99a and miR-100 in cases resistant to vincristine or daunorubicin. In conclusion, genetic subtypes and drug resistant leukemic cells display characteristic miRNA expression levels. Functional studies are indicated for discriminative miRNAs and may provide new insights into leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Disturbed CXCR4/CXCL12 Axis In Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.2643.2643 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2643-2643 ◽

Cited By ~ 1

Author(s):

Lieke C.J. van den Berk ◽

Arian van der Veer ◽

Marieke E. Willemse ◽

Myrte J.G.A. Theeuwes ◽

Mirjam W. Luijendijk ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Serum Levels ◽

Initial Diagnosis ◽

Leukemic Cells ◽

Cxcr4 Receptor ◽

Cell Acute Lymphoblastic Leukemia

Abstract Malignant cells that infiltrate the bone marrow (BM) interfere with the normal cellular behavior of supporting cells, thereby creating an alternative malignant niche. This intercellular communication is mostly mediated by cytokines and their receptors. In this study, we find that expression of the CXCR4 receptor is significantly increased in pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) cells compared with normal mononuclear hematopoietic cells derived of the bone marrow (p=0.016). Furthermore, we show that high CXCR4 expression is correlated with an unfavorable clinical outcome in BCP-ALL (5-yr CIR ±SE: 38.4% ±6.9% in CXCR4-high versus 12.0% ±4.6% in CXCR4-low expressing patients, p<0.001). Interestingly, BM serum levels of the CXCR4 ligand (CXCL12) are 2.7-fold lower (p=0.005) in samples taken at initial diagnosis of BCP-ALL compared with the levels in samples taken of non-leukemic controls. We show that induction chemotherapy restores CXCL12 levels in the BM to normal levels. Blocking the CXCR4 receptor with Plerixafor (FDA-approved drug) showed that the lower CXCL12 serum levels at initial diagnosis could not be explained by consumption by the leukemic cells, nor did we observe an altered CXCL12-production capacity of BM-MSC at this time-point. We rather observed that a very high density of leukemic cells negatively affected CXCL12 production by the BM-MSC while stimulating the secretion levels of G-CSF. These results suggest that highly proliferative leukemic cells are able to down-regulate the production of cytokines involved in homing (CXCL12), while simultaneously up-regulating the production of cytokines involved in hematopoietic mobilization (G-CSF). This disbalance may stimulate the spreading of BCP-ALL outside the BM. The data presented here suggest that interference with the CXCR4/CXCL12 axis (for instance by using Plerixafor) may be an effective way to mobilize BCP-ALL cells; the more ALL cells become mobilized, the less ALL cells may escape from combination chemotherapy. In proof-of concept studies, this hypothesis needs to be validated to pave the way for implementation in future treatment protocols for children with ALL. Disclosures: No relevant conflicts of interest to declare.

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B-Cell Precursor Acute Lymphoblastic Leukemia Cells Create a Self-Reinforcing Niche Independent of the CXCR4/CXCL12 Axis

Blood ◽

10.1182/blood.v126.23.1297.1297 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1297-1297

Author(s):

Bob de Rooij ◽

Roel Polak ◽

Rob Pieters ◽

Monique L. Den Boer

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Leukemic Cells ◽

Cell Precursor ◽

Cxcr4 Antagonist

Abstract Background Acute lymphoblastic leukemia (ALL) cells create a leukemic niche that protects malignant cells from the effects of cytostatic agents and immune cells by altering their bone marrow microenvironment. This malignant process can be counteracted by impairing the homing of leukemic cells towards the bone marrow. Hematopoietic cells express the chemokine receptor CXCR4 and migrate towards its ligand CXCL12, which is actively produced by MSCs in the bone marrow. Therefore clinical trials have been initiated using the CXCR4 antagonist AMD3100 (Plerixafor) during leukemia treatment. However, these trials, as well as priming of AML in more than 4000 patients using a CXCR4 dependent mechanism, have not resulted in improved overall survival rates. This suggests that CXCR4 inhibition is not sufficient to disrupt leukemic niches. Objectives In this study we investigated how leukemic cells regulate the chemoattractive properties of their microenvironment. Results Here we show, using an ex vivo niche model with primary MSCs, that B-cell precursor ALL (BCP-ALL) cells affect their healthy microenvironment without altering CXCL12 secretion. Using a transwell migration assay we studied the chemoattractive properties and chemokine secretion patterns of several cell types and co-cultures. We confirmed that BCP-ALL cells migrate towards a CXCL12 gradient produced by primary MSCs (11-fold more migrated cells compared to background, p < 0.001). Inhibition of CXCR4 by AMD3100 reduced migration towards MSCs by 80% (p < 0.01). BCP-ALL cells migrated even more towards co-cultures of BCP-ALL cells and primary MSCs (24-fold more migrated cells compared to background, p < 0.001). Strikingly, this ex vivo leukemic niche did not produce higher levels of CXCL12 compared to MSC mono-cultures. Moreover, the induced migration towards MSC-ALL co-cultures could not be inhibited by AMD3100 treatment, indicating that BCP-ALL cells enhance the chemoattractive properties of their microenvironment in a CXCL12-independent manner. In contrast to BCP-ALL cells, the migration of CD34+ progenitor cells towards co-cultures of BCP-ALL cells and MSCs was significantly reduced (0.8-fold compared to migration towards MSCs, p < 0.05). Similar results were observed when we studied the migratory behavior of MSCs. MSCs actively migrated towards BCP-ALL cells (1.7 fold compared to background, p < 0.001), while migration of MSCs was significantly reduced towards MSC-ALL co-cultures (0.4-fold compared to migration towards BCP-ALL, p < 0.001). To find candidate factors influencing this process, we quantified the secreted levels of 64 cytokines in co-cultures of patient-derived BCP-ALL cells and MSCs. We observed leukemia-driven cytokine secretion patterns that were not influenced by the source of primary MSCs. In contrast to unaltered levels of CXCL12, we observed significant inductions of MCP-1/CCL2 and MDC/CCL22 (CCR4-ligands), IL8 and GRO-1 (CXCR1/2-ligands) and IP10/CXCL10 (CXCR3-ligands). Conclusion Our data indicate that leukemic cells alter the chemoattractive properties of their microenvironment, resulting in the secretion of multiple chemokines into the leukemic niche. This leukemic niche is highly potent in attracting BCP-ALL cells and repels the influx of healthy hematopoietic cells and MSCs using a CXCL12-independent mechanism. Furthermore, our results identify candidate factors that might be valuable future therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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