scholarly journals Implication of ICOSLG on Relapse in Infant T(4;11) Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3481-3481
Author(s):  
Marius Külp ◽  
Anna Lena Siemund ◽  
Claus Meyer ◽  
Patrizia Larghero ◽  
Alissa Dietz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Therapeutic Target ◽  
Lymphoblastic Leukemia ◽  
Sleeping Beauty ◽  
Immune Privilege ◽  
Immune Recognition ◽  
Dependent Manner

Abstract Infant t(4;11) acute lymphoblastic leukemia (ALL) is associated with a high relapse rate with 4-year event-free survival (EFS) of only 36%. Relapse has been shown to be the major cause of death as 83% of relapsed infant t(4;11) ALL patients die within three years of diagnosis. Therefore, it is of utmost therapeutic interest to elucidate molecular mechanisms of relapse. Here we show that t(4;11) ALL cells upregulate the inducible T-cell costimulator ligand (ICOSLG) in an early growth response 3 (EGR3) dependent manner thereby promoting the development of regulatory T-cells (T regs). We propose that short EFS and high ICOSLG expression are causally linked, in that ICOSLG-mediated induction of T regs interferes with immune recognition of leukemia cells. According to that hypothesis, ICOSLG would not only be a novel and independent prognostic biomarker but a potential therapeutic target. We investigated the function of EGR3 in infant ALL since EGR3 has been described as an indirect target of the iroquois homeobox 1 (IRX1) transcription factor. For that purpose we created a stable sleeping beauty transposon-based SEM cell line expressing EGR3 in a Doxycycline-inducible manner. Gene expression and western blot analysis revealed strong upregulation of ICOSLG 48h after Doxycycline induction when compared to an empty vector control. Additionally, chromatin immunoprecipitation (ChIP) experiments depicted that EGR3 directly binds to the promoter of the ICOSLG gene. The expression of ICOSLG in mesenchymal stem cells has been shown to foster the induction of T regs and ICOSLG-mediated T reg expansion has been identified as a driver of acute myeloid leukemia and glioblastoma. In the bone marrow (BM) hematopoietic stem cells (HSC) colocalize with T regs which provide an immune privilege to the stem cell niche. Based on these observations, we hypothesize that ICOSLG expressing ALL cells could create an immune privileged niche appearing to be necessary for HSC maintenance and sanctuary from immune attack. This could create independence from the BM immune privilege enabling migration of the ALL cells. If so, this could contribute to minimal residual disease (MRD) formation after induction therapy and subsequently to higher probability of relapse. To evaluate our hypothesis, we cocultured the EGR3-SEM and control cells with primary human CD4 + and CD8 + T-cells. The T-lymphocytes were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors and stimulated with coated α-CD2, -CD3 and -CD28 beads. We observed 16% more CD4 +CD25 ++FOXP3 + T regs after 48h of coculture with EGR3-SEM cells compared to the control. The addition of a neutralizing monoclonal α-ICOSLG antibody to the coculture led to a reduction of the T reg frequency in EGR3-SEM coculture. Taken together, these results strongly suggest that ALL cells expressing EGR3 induce the formation of T regs via ICOSLG expression. To confirm our results with patient-derived material, we investigated the gene expression of 50 infant t(4;11) pro-B phenotypic ALL patients. Pearson correlation testing confirmed that ICOSLG expression strongly correlates with EGR3 and IRX1 expression. Furthermore, we were able to classify the patients considering their ICOSLG expression level into an ICOSLG-high (ICOSLG-hi) and an ICOSLG-low (ICOSLG-lo) group. Outcome data for a 5-year follow-up were available for 35 of 50 patients, n=5 ICOSLG-hi and n=30 ICOSLG-lo. 100% (5/5) of the ICOSLG-hi patients failed within 17 months of diagnosis whereas 53% (16/30) of the ICOSLG-lo patients failed within 56 months of diagnosis. The remaining 14 ICOSLG-lo patients were alive after 60 months. These data underscore the role of overexpressed ICOSLG in relapse development. However, a verification of these findings in a larger cohort is needed. In conclusion, our study identifies ICOSLG as a promising prognostic marker and novel therapeutic target in infant t(4;11) ALL. Furthermore, our findings implicate the interaction between T-cells and leukemia stem cells as contributory to disease progression. Disclosures Cario: Novartis: Other: Lecture Fee.

KLF4 Acts As a Tumor Suppressor in T-Acute Lymphoblastic Leukemia and Negatively Regulates Human T Cell Development and Homeostasis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2405-2405
Author(s):  
Bing Xu ◽  
Peng Li
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Cell Development ◽  
Dependent Manner ◽  
Human T Cell

Abstract The transcription factor Kruppel-like factor 4 (KLF4) may induce tumorigenesis or suppress tumor growth in a tissue-dependent manner. We found that overexpression of KLF4 induced not only human acute T-acute lymphoblastic leukemia (T-ALL) cell lines but also primary samples from T-ALL patients to undergo apoptosis through the BCL2/BCLXL pathway in vitro. T cell-associated genes including BCL11B, GATA3, and TCF7 were negatively regulated by KLF4 overexpression. Especially, KLF4 induced SUMOylation and degradation of BCL11B. However, the KLF4-induced apoptosis in T-ALL was rescued by the in vivo microenvironment. Furthermore, the invasion capacity of T-ALL to hosts was compromised when KLF4 was overexpressed. In normal human T cells, the overexpression of KLF4 severely impaired T cell development at early stages, but the blockage of T cell development was resumed by restoration of GATA3 or ICN1. In summary, our data demonstrate that KLF4 acts as a tumor suppressor in malignant T cells and that downregulation of KLF4 may be a prerequisite for early human T cell development and homeostasis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cell of origin strongly influences genetic selection in a mouse model of T-ALL

Blood ◽  
2011 ◽  
Vol 118 (17) ◽  
pp. 4646-4656 ◽  
Author(s):  
Katherine E. Berquam-Vrieze ◽  
Kishore Nannapaneni ◽  
Benjamin T. Brett ◽  
Linda Holmfeldt ◽  
Jing Ma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Genetic Selection ◽  
Sleeping Beauty ◽  
Cell Of Origin ◽  
Human T Cell ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Identifying the normal cell from which a tumor originates is crucial to understanding the etiology of that cancer. However, retrospective identification of the cell of origin in cancer is challenging because of the accumulation of genetic and epigenetic changes in tumor cells. The biologic state of the cell of origin likely influences the genetic events that drive transformation. We directly tested this hypothesis by performing a Sleeping Beauty transposon mutagenesis screen in which common insertion sites were identified in tumors that were produced by mutagenesis of cells at varying time points throughout the T lineage. Mutation and gene expression data derived from these tumors were then compared with data obtained from a panel of 84 human T-cell acute lymphoblastic leukemia samples, including copy number alterations and gene expression profiles. This revealed that altering the cell of origin produces tumors that model distinct subtypes of human T-cell acute lymphoblastic leukemia, suggesting that even subtle changes in the cell of origin dramatically affect genetic selection in tumors. These findings have broad implications for the genetic analysis of human cancers as well as the production of mouse models of cancer.

ZMIZ1 Collaborates with NOTCH1 During Induction and Maintenance of T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 75-75
Author(s):  
Lesley A Rakowski ◽  
Derek D Garagiola ◽  
Sarah C Caruso ◽  
Mark Y. Chiang
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Potential Therapeutic Target ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Notch1 Mutations

Abstract Abstract 75 Activating NOTCH1 mutations are found in 40–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, most leukemia-associated NOTCH1 mutations fail to induce leukemia. This observation suggests that cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. In murine retroviral/transposon insertional mutagenesis screens, induction of the Zmiz1 gene was frequently associated with activation of the Notch1 receptor during leukemogenesis (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT-like family that has been implicated for prostate cancer survival. It directly interacts with the androgen receptor to enhance its transcriptional activity. To investigate the role of ZMIZ1 during leukemogenesis, activating NOTCH1 mutations and ZMIZ1 were transduced into hematopoietic progenitor cells. These cells were then used to reconstitute lethally irradiated mice. ZMIZ1 or NOTCH1 alone failed to induce T-ALL after 1 year of observation. In contrast, ZMIZ1 and NOTCH1 in combination induced T-ALL with ∼50% penetrance by 100 days after transplantation. These data show that ZMIZ1 can promote leukemogenesis in cooperation with NOTCH1. To determine the relevance of ZMIZ1 to human leukemia, we screened 15 primary human adult T-ALL samples for ZMIZ1 mRNA and protein. 20% expressed ZMIZ1. In publically available data sets, ZMIZ1 gene expression was significantly enriched by ∼2-fold in early thymocyte precursor ALL (ETP-ALL) samples. The ETP-ALL subgroup comprises about 13% of all T-ALL and may have a highly unfavorable prognosis. We discovered expression of ZMIZ1 in two ETP-like cell lines. To investigate whether ZMIZ1 is a potential therapeutic target, we transduced these cell lines with shRNA directed against ZMIZ1. ZMIZ1 inhibition reduced cell size, increased apoptosis by ∼2-fold, and reduced growth by 75–94%. Furthermore, ZMIZ1 knockdown overcame resistance to NOTCH signaling blockade with g-secretase inhibitors. Since the glucocorticoid receptor is highly homologous to the androgen receptor, we considered the possibility that ZMIZ1 inhibition may promote glucocorticoid resistance. However, we treated ZMIZ1-inhibited T-ALL cell lines with increasing doses of dexamethasone. ZMIZ1-inhibited T-ALL cell lines were twice as sensitive to dexamethasone than uninhibited cells. These data suggest that ZMIZ1 is required for leukemia growth and survival. Inhibition of ZMIZ1 may potentially enhance targeting of T-ALL with NOTCH pathway inhibitors and glucocorticoids. To determine the mechanism underlying ZMIZ1 function, we performed gene expression profiling. We identified C-MYC as a potential downstream target of ZMIZ1. C-MYC is also a direct target of NOTCH1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous C-MYC expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by ∼7000 fold, induced C-MYC target gene expression, and rescued the C-MYC dependent cell line. ZMIZ1 inhibition lowered C-MYC levels by ∼93%. The interaction between ZMIZ1 and NOTCH appeared to be specific for C-MYC, as modulation of ZMIZ1 levels did not affect the NOTCH1 target genes Hes1, Dtx1, and Cd25. Downregulation of C-MYC partly phenocopied the effects of ZMIZ1 downregulation. However, ectopic expression of C-MYC failed to rescue the growth of ZMIZ1-inhibited cells. These data suggest that C-MYC is an essential but insufficient downstream effector of ZMIZ1 function. In conclusion, ZMIZ1 is new potential therapeutic target in a subset of T-ALL. It functionally interacts with NOTCH1 to promote C-MYC expression and activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Donor-Derived CAR T Cells Engineered with Sleeping Beauty in Pediatric and Adult Patients with Acute Lymphoblastic Leukemia Relapsed Post-HSCT

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 472-472
Author(s):  
Chiara F Magnani ◽  
Giuseppe Gaipa ◽  
Federico Lussana ◽  
Giuseppe Gritti ◽  
Daniela Belotti ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Dose Level ◽  
Dose Escalation ◽  
Lymphoblastic Leukemia ◽  
Sleeping Beauty ◽  
Advisory Board ◽  
Adult Patients ◽  
Car T Cells ◽  
Car T

Abstract Introduction Allogeneic Chimeric Antigen Receptor (CAR) T cells engineered with non-viral methods offer a modality to reduce costs and logistical complexity of the viral process and allow lymphodepleted patients to access CAR T cell treatment. We recently proposed the use of Sleeping Beauty (SB) transposon to engineer donor-derived T cells differentiated according to the cytokine-induced killer (CIK) cell protocol (Magnani CF et al. J Clin Invest. 2021). We report here outcomes on B-cell acute lymphoblastic leukemia (B-ALL) patients, relapsing after transplantation, treated with donor-derived anti-CD19 CAR T cells (CARCIK-CD19). Methods We conducted an academic, multi-center, phase I/II dose-escalation trial in patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The infusion product was manufactured in-house starting from 50 mL of peripheral blood from the HSCT donor by electroporation with GMP-grade plasmids. All patients underwent lymphodepletion with Fludarabine (30 mg/m 2/day x 4 days) and Cyclophosphamide (500 mg/m 2/day x 2 days), before proceeding to CARCIK-CD19 infusion. We used the Bayesian Optimal Interval (BOIN) design to define a four-dose escalation scheme. Primary objectives were to define the Maximum Tolerated Dose (MTD), safety, and feasibility. Secondary objectives included the assessment of complete hematologic response (CR), duration of response (DOR), progression-free (PFS), event-free (EFS), and overall survival (OS). This study was registered at ClinicalTrials.gov, NCT03389035. Results From January 2018 to June 2021, a total of 32 patients were screened, 26 enrolled (6 children and 20 adults) and 21 infused (4 children and 17 adults). Reasons for not receiving infusion included consent withdrawal (N=1), disease progression not controlled by bridging therapy (N=3), acquisition of myeloid phenotype (N=1). The median number of prior therapies was 4 (range, 1-7) with a median time interval from HSCT to relapse of 9 months. The median BM blasts was 60% (range, 5-100%) at enrollment and 7% (range, 0-96%) post lymphodepletion. Of the 21 patients infused, CARCIK-CD19 were obtained by HLA-identical sibling (n=6, 29%), matched unrelated (n= 7, 33%), and haploidentical donors (n=8, 38%). Three patients (14%) received the first dose level of 1x10 6 CARCIK-CD19 cells/Kg, three (14%) the second of 3x10 6, and three (14%) the third of 7.5x10 6 whereas 12 patients (57%) received the fourth and last planned dose level of 15x10 6 cells/Kg, as no dose limiting toxicity (DLT) was observed. CRS was observed in six patients (three grade I and three grade II) and immune effector cell-associated neurotoxicity in two patients at the highest dose. Although 9 out of 21 had experienced acute or chronic graft-versus-host disease (GvHD) after the previous HSCT, secondary GvHD was never induced by CARCIK-CD19. Complete response was achieved by 13 out of 21 patients (61.9%, 95%CI=38-82%) and by 11 out of 15 patients treated with the 2 highest doses (73.3%, 95%CI=45-92%). Eleven of these responders were MRD-negative. Notably, the type of donor did not influence the achievement of CR 28 days post-infusion. At a median follow up of 21.6 months (range, 1.0-38.4 months), 10 patients (47.6%) are alive in CR (9 in the 2 highest dose levels). Overall, the median OS and EFS were 9.7 and 3.2 months, respectively, with a median DOR of 4.0 months (range, 1.0-23.5 months). Patients in CR at 28-days had a 6-months relapse-free survival of 48.4% (SE=14.9). EFS at 6 months was 26.5% (SE=9.9) and OS was 67.6% (SE=11.1). Among the 13 patients who achieved CR, two children underwent consolidation with a second allo-HSCT in complete remission. Adult patients did not receive any additional anti-leukemic therapies unless a relapse occurred, and four of them remained in remission and alive (+24, +9, +6, and +4 months). Robust CARCIK-CD19 cell expansion was achieved in most patients and CARCIK-CD19 cells were measurable for up to 22 months. Conclusions SB-engineered CAR T cells induce sustained responses in B-ALL patients relapsed after HSCT irrespective of the donor type and without severe toxicities. Disclosures Lussana: Incyte: Honoraria; Pfizer: Honoraria; Astellas Pharma: Honoraria; Amgen: Honoraria. Gritti: Takeda: Consultancy; Roche: Consultancy; Kite Gilead: Consultancy; IQvia: Consultancy; Italfarmaco: Consultancy; Clinigen: Consultancy. Biondi: Incyte: Consultancy, Other: Advisory Board; Bluebird: Other: Advisory Board; Novartis: Honoraria; Amgen: Honoraria; Colmmune: Honoraria.

scholarly journals Genomic Determinants of Response to Blinatumomab in Relapsed/Refractory (R/R) B-Cell Precursor Acute Lymphoblastic Leukemia in Adults

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1552-1552 ◽  
Author(s):  
Yaqi Zhao ◽  
Ibrahim Aldoss ◽  
Chunxu Qu ◽  
Guido Marcucci ◽  
Anthony S. Stein ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
B Cell ◽  
High Frequency ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
B Lymphoid

Abstract Introduction: Blinatumomab, a CD19/CD3 bi-specific T-cell engager monoclonal antibody that re-directs CD3-positive T cells towards CD19-positive B cells, has shown promise in the treatment of R/R B-cell precursor acute lymphoblastic leukemia (B-ALL), with superior survival rates compared to salvage chemotherapy. However, many patients do not respond or subsequently relapse, and the mechanisms underlying resistance are unclear. The goals of this study were to characterize the genomic features associated with response to blinatumomab. Methods: We studied 29 patients (pts; median age 28, range 18-70) with R/R B-ALL who were treated with up to 5 cycles of blinatumomab, and predominantly of Hispanic ancestry (66%). Overall, 17 pts (59%) achieved remission with blinatumomab whilst 12 showed no response. Among the 17 responders, 7 (41%) subsequently relapsed or progressed during treatment. We analyzed leukemic blasts obtained before and after blinatumomab treatment whenever available: pre-blinatumomab R/R (n=28), post-blinatumomab refractory (n=5), post-blinatumomab relapsed (n=3) (Figure 1). Leukemia and matched remission samples were studied using transcriptome sequencing (n=34), whole genome sequencing (n=28), whole exome sequencing (n=19) and Infinium Omni2.5Exome-8 (SNP array, n=19). Results: Seventeen of 29 pts (59%) were Ph-like ALL. Twelve of 17 Ph-like ALL pts had high CRLF2 expression, among these we identified P2RY8-CRLF2 (n=4) and IGH-CRLF2 (n=8). Within the remaining 5 Ph-like ALL cases, two pts harbored NUP214-ABL1, two IGH-EPOR and one TERF-JAK2. Fifteen of the 17 (88%) Ph-like ALL cases were of Hispanic ancestry. The prevalence of other known subtypes was relatively low: BCR-ABL1 7%, hypodiploid 7%, KMT2A 3%, TCF3-PBX1 3% and B-other 21%. We observed a high response rate of 83.3% (10/12 cases) in Ph-like_CRLF2 pts, whilst the frequency of response was 60% (3/5, including two IGH-EPOR) for Ph-like_non-CRLF2 pts, and 33% (4/12) for the other subtypes (Ph-like ALL vs. others, P=0.029) (Figure 1). Unsupervised hierarchical clustering of pre-blinatumomab samples identified 3 clusters based on response to blinatumomab: cluster 1 contained non-responders, clusters 2 and 3 were largely made up of responders. By gene expression profiling using CIBERSORT we found reduced infiltration of cytotoxic CD8+ T-cells in cluster 1 compared to clusters 2 and 3 (6.1% vs. 14.9%, P=0.014), which was inversely correlated with the presence of CD4+ T cells (17.9% vs. 11.5%). GSEA showed enrichment for the IFNγ response, JAK-STAT signaling, chemokine and cytokine signaling in responders. In non-responders, differential gene expression analysis identified up-regulation of the H3K4 demethylase KDM5B, an oncogene associated with progression and chemoresistance of glioma and neuroblastoma. We observed a high frequency of alterations affecting B-lymphoid development (IKZF1, PAX5 and EBF1) in the pre-blinatumomab samples (20 of 22, 91%), which were maintained during progression or relapse. The frequency of B-lymphoid alterations did not differ significantly between responders and non-responders (13 of 14, 93% vs. 7 of 8, 88%). Alterations affecting the cell cycle (CDKN2A/B, TP53, RB1) were observed at a high frequency in pre-blinatumomab samples (15 of 22, 68%), with CDKN2A/B deletions enriched in responders compared to non-responders (11 of 14, 79% vs. 2 of 8, 25%; P=0.026). We also observed a high prevalence of alterations affecting epigenetic modifiers (ARID1B, CREBBP, KDM6A, KMT2D, TRRAP, SMARCA4) in pre-blinatumomab samples (17 of 22, 77%), with no difference between responders and non-responders (10 of 14, 71% vs. 7 of 8, 88%; P=0.61). Of the post-blinatumomab R/R samples available for study (n=8), CD19 expression was negative (n=1), dim (n=2) or positive (n=5). In contrast to previous reports of CD19 escape in CAR T-cell treated patients, there was no evidence of aberrantly spliced CD19 mRNA species, CD19 mutation or deletion in the three negative/dim cases. Conclusion: We show that a heightened immune response through the infiltration of cytotoxic T-cells and activation of IFNγ and JAK-STAT signaling in leukemic cells is an important determinant of response to blinatumomab. Importantly, blinatumomab is a valid therapeutic approach for patients harboring high-risk CRLF2 and EPOR-rearrangements. CD19 escape is not associated with genetic alterations at the CD19 locus. Figure. Figure. Disclosures Stein: Amgen Inc.: Speakers Bureau; Celgene: Speakers Bureau. Mullighan:Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

ANGIOPOIETIN1 - a Novel Factor Implicated In MLL-Rearranged Acute Lymphoblastic Leukemia and Regulated In a Fusion Gene-Dependent Manner

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 66-66 ◽  
Author(s):  
Patricia Garrido Castro ◽  
Simon Bomken ◽  
Lidija Seslija ◽  
Ronald Stam ◽  
Elda S Latif ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Lymphoblastic Leukemia ◽  
Dependent Manner ◽  
Qrt Pcr

Abstract Abstract 66 Background: MLL-rearranged acute lymphoblastic leukemia (ALL) is prevalent in infants, constituting 70% of the cases. The preferred MLL translocation partner is the gene AF4, resulting in t(4;11)(q21;q23), which arises in 50% of infant ALL patients. This translocation generates the fusion genes MLL/AF4 and AF4/MLL, and is associated with an aggressive clinical presentation and poor outcome. Biologically, cells expressing MLL/AF4 show resistance to stress- and chemotherapy-related apoptosis. Concordantly, we have previously shown that RNAi-mediated depletion of MLL/AF4 in the t(4;11)-positive ALL cell line SEM results in induction of cell death and impaired both clonogenicity and in vivo engraftment. In order to characterize this phenotype on a molecular level, we have performed gene expression profiling of SEM cells depleted of MLL/AF4 and corresponding controls. Expression of >1000 genes was affected, including a subset of angiogenic genes, most prominently ANGIOPOIETIN1 (ANGPT1), a proangiogenic cytokine reported to play a role in acute myeloid leukemia (AML), hematopoietic stem cell (HSC) quiescence and bone marrow (BM) niche maintenance, but to date not implicated in ALL. Here we report a novel link between ANGPT1 expression and MLL-rearranged ALL. Methods: Gene expression profiling was performed using the Illumina HT-12 platform and data processed using BeadStudio and Genespring software suites. ANGPT1 expression was analyzed by real-time RT-PCR (qRT-PCR), and ANGPT1 protein secretion determined using enzyme-linked immunosorbent assay (ELISA). The MLL/AF4 status of cells was modulated with fusion transcript-specific siRNAs and knockdown monitored by qRT-PCR. RNAi-mediated depletion of ANGPT1 was achieved using siRNA or lentiviral shRNA constructs, and validated on transcript and protein level. Effects on cell cycle progression and proliferation in response to ANGPT1 knockdown in t(4;11)-positive cells were assessed by flow cytometry and trypan blue exclusion assay, respectively. For in vivo studies, SEM cells were sequentially transduced to express both luciferase and either non-target control shRNA (shNTC) or shANGPT1. Doubly transduced cells were selected for and FACS-sorted prior to intrafemoral transplantation into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Disease progression was monitored using bioluminescence imaging and engraftment assessed by flow cytometry at the terminal timepoint. Results: ANGPT1 expression was screened in a MLL-rearranged ALL patient cohort (n=35), comprising t(4;11)-positive (n=20), t(11;19)-positive (n=10) and t(9;11)-positive patients (n=5), and in a MLL-wildtype BCP ALL patient cohort (n=8). MLL-rearranged patients showed ANGPT1 upregulation, t(4;11)-positive patients having the strongest overexpression by 232-fold when compared to ANGPT1 levels in CD19+ peripheral blood (PB) cells. A 27-fold and 13-fold upregulation was detected in t(11;19)- and t(9;11)-positive patients, respectively. Conversely, MLL-wildtype BCP ALL patients had similar ANGPT1 levels as CD19+ PB cells, with only a 2-fold increase. In addition to its high expression in t(4;11)-positive ALL, ANGPT1 levels were shown to be dependent on MLL/AF4; a reduction of ANGPT1 mRNA and protein correlated with siRNA-mediated MLL/AF4 depletion in a time-dependent manner in both cell lines and primary patient samples. This was concordant with expression array data, which indicated an up to 4-fold decrease of ANGPT1 in response to MLL/AF4 depletion. The functional role of ANGPT1 in t(4;11)-positive ALL was assessed by RNAi; sustained depletion of ANGPT1 in SEM cells resulted in cell cycle arrest and a marked decrease in proliferation. In vivo, mice transplanted with shANGPT1 expressing SEM cells showed reduced splenic infiltration and development of solid tumours at the injection site, as opposed to a systemic spread of the disease and massive splenomegaly in mice injected with shNTC expressing SEM cells. Conclusions: In this study we have identified ANGPT1 as a novel player in t(4;11)-positive ALL, as defined by overexpression, MLL/AF4-dependent regulation and functional consequences in vivo and in vitro. Currently we are investigating ANGPT1-mediated signalling in t(4;11) ALL cells, as it represents an attractive potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Mir∼17-92 Identifies BCL2 As a Therapeutic Target In BCR-ABL Positive B-Lineage Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 835-835
Author(s):  
Matthias Eder ◽  
Alex Elder ◽  
David Barzan ◽  
Karin Battmer ◽  
Simon Bomken ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Isotope Labeling ◽  
Lymphoblastic Leukemia ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Over Expression ◽  
Specific Manner

Abstract Despite advances in both targeted therapies with ABL-specific tyrosine kinase inhibitors and in allogeneic stem cell transplantation, BCR-ABL positive acute lymphoblastic leukemia (ALL) remains a very high-risk disease, necessitating the development of novel treatment strategies. miRNAs are small non-coding RNAs which regulate gene expression posttranscriptionally in a sequence-specific manner. miRNAs usually repress the expression of many target genes. We hypothesized that miRNAs may help to identify potential therapeutic targets if (i) they are expressed in a disease-specific manner and if (ii) modulating their expression induces a desired phenotype, such as apoptosis of tumour cells, in appropriate experimental models. Based on our observation that miR∼17-92-encoded miRNAs are significantly less abundant in primary BCR-ABL-positive as compared to negative ALL-cells, we studied the expression and function of miRNAs encoded by the miR∼17-92 derivative miR∼17-19b in a murine pro-B-cell line with inducible BCR-ABL-expression (TonB). Induction of BCR-ABL expression in TonB cells reduced endogenous miR-17, miR-18a, and miR-19 by 2 to 3.5-fold, confirming that expression of the miR∼17-92 cluster is controlled by BCR-ABL. Interestingly, over-expression of miR∼17-19b by lentiviral gene transfer led to a substantial induction of apoptosis in TonB cells in a BCR-ABL-dependent manner. To identify potential miRNA targets, we used a proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) in miR∼17-19b transgenic TonB cells. Several apoptosis-related proteins were differentially expressed including Bcl2, an established inhibitor of mitochondrial pro-apoptotic pathways. The miRNA target prediction program RNA22 predicted several miR∼17-19b miRNA-binding sites within both murine and human Bcl2 mRNA, and we demonstrated direct miRNA binding to Bcl2 mRNA by luciferase reporter and anti-AGO2 RIP chip analyses. As with miR∼17-19b over-expression, Bcl2 specific RNAi strongly induced apoptosis in murine TonB and the human BCR-ABL-positive cell lines BV-173, Tom1 and SupB15. BCR-ABL positive human ALL-cell lines were also more sensitive than negative ones to pharmacological BCL2 inhibition with the BH3 mimetic ABT 737. In addition, inhibition of BCL2 by ABT 737 and BCR-ABL kinase activity by Imatinib exert different anti-leukemic effects with differential impact on miR∼17-92 miRNA-expression. To assess the therapeutic potential of BCL2 inhibition we used a xenotransplantation assay with real time in vivo monitoring of drug therapies by bioluminescent imaging. ABT-737 treatment substantially inhibited expansion of luciferase-expressing human primary BCR-ABL-positive ALL xenografts in NOD/LtSz-scid IL-2Rγ null (NSG) mice and significantly lengthened their median survival. Taken together, our data identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL and indicate involvement of miR∼17-92-encoded miRNAs in regulation of apoptosis in these cells. The validity of this miRNA-based approach to identify potential drug targets is demonstrated by the efficacy of the BCL2 inhibitor ABT-737 in an in vivo model of human BCR-ABL positive ALL, suggesting that BCL2 inhibition should be considered for early phase clinical testing as a strategy to improve disease outcomes. Disclosures: No relevant conflicts of interest to declare.

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