An Azaindole-Based Small Molecule Hzx-02-059 Induces Methuosis in B-Cell Acute Lymphoblastic Leukemia through the PI3K/AKT Axis

Methuosis is a newly defined nonapoptotic cell death characterized by the accumulation of large liquid-filled vacuoles in cytoplasm, which is distinct from necrosis, autophagy and apoptosis. A novel azaindole-based methuosis inducer, named HZX-02-059, displays cytotoxic effects to a series of cancer cellular models via blocking the role of PIKfyve. The discovery of HZX-02-059 sheds light on a novel therapeutic option for refractory cancer patients who have limited effective treatments. Despite significant advances in treatments, over 50% of adult patients with B-cell acute lymphoblastic leukemia (B-ALL) fail to achieve durable responses with current intensive chemotherapies, which highlight an urgent need of new therapeutic regimens for this patient population. Here, we applied HZX-02-059 to B-ALL cell lines and mice models to uncover its potential role and underlying mechanism. Morphology changes of HZX-02-059-treated B-ALL cells were observed under the light microscope, and these treated cells were to certain extent collapsed into debris. Of note, higher concentration of this compound induced the accumulation of substantial larger vacuoles in cytoplasm. To assess the cytotoxicity of this compound, we performed cell counting kit-8 (CCK-8) and colony forming unit (CFU) assays. As expected, HZX-02-059 significantly inhibited cancer cell proliferation and reduced the capability of cell colony forming in a dose-dependent manner. Next, we carried out an EdU assay to determine the influence of HZX-02-059 on cell cycle distribution. As a result, B-ALL cells treated with HZX-02-059 were mainly blocked at G2/M phase. Western blotting was conducted to investigate the underlying molecular basis of HZX-02-059 in B-ALL. To the end, we found that HZX-02-059 negatively regulated the proteins involved in the PI3K/AKT pathway. Also, this drug decreased the expression of the pathway downstream transcription factors NF-ǐB and c-Myc, suggesting that the dysregulation of the PI3K/AKT axis is a potential mechanism of action of HZX-02-059 against B-ALL. To further evaluate itsin vivotherapeutic effect, B-ALL patient-derived xenograft (PDX) mice were established and administrated with HZX-02-059. The result revealed a survival benefit of the treatment as compared with the control, which grants this molecule great potential for clinical usage. Thus, our results suggest that HZX-02-059 is a potent anti-BALL reagent in preclinical models that warrant further investigation of this drug as an alternative therapeutic strategy for B-ALL patients who are resistant to conventional therapies. Disclosures No relevant conflicts of interest to declare.

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IKZF1deletions Coupled with CD20 Expression Represents a Novel High-Risk Subtype in Adult B-Cell Progenitor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-154271 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4474-4474

Author(s):

Bingqing Tang ◽

Zhixiang Wang ◽

Dainan Lin ◽

Xianjun He ◽

Zihong Cai ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

High Risk ◽

B Cell ◽

Validation Cohort ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Hematopoietic Stem ◽

Poor Outcome ◽

Cd20 Expression ◽

Cell Acute Lymphoblastic Leukemia

Abstract Genetic deletions of IKZF1 are associated with poor prognosis in B-cell acute lymphoblastic leukemia (B-ALL). Here we investigated the effect of IKZF1 deletions (IKZF1 del) plus with immunotype in adult B-ALL in PDT-ALL-2016 cohort. This cohort study involved 161 patients with B-ALL from 2016 to 2019, with detailed information about IKZF1 del and CD20 expression. Validation cohort consists N= patients from TARGET cohort. IKZF1 del was detected in 36.0% of patients with 3-year event-free survival (EFS) of 37.2±6.7% and overall survival (OS) of 51.1±7.3%, compared to IKZF1 wild-type (IKZF1 wt) with EFS 55.4±5.1% (P<0.01) and OS 74.6±4.5% (P<0.05), respectively. CD20 expression was also associated with inferior EFS than CD20-negative group (P<0.05). Furthermore, IKZF1 del coupled with CD20 expression, termed as IKZF1 del/CD20+, comprised 12.4% of patients with 3-year EFS of 25.0±9.7% compared with IKZF1 wt (P<0.05 ) and IKZF1 del/CD20- (P<0.05 ) groups, respectively. Multivariable analyses demonstrated independence of IKZF1 del/CD20+ with highest hazard ratio for EFS and OS. Furthermore, the prognostic strength of IKZF1 del/CD20+ was confirmed in TARGET validation cohort. Eighty-one patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Notably, neither IKZF1 del(P=0.6288), CD20 (P=0.0705) or IKZF1 del/CD20 (P=0.3410) groups were identified as poor outcome in allo-HSCT cohort. Collectively, our data demonstrate that IKZF1 del/CD20+ represents a very high-risk subtype in adult B-ALL; and particularly, allo-HSCT could overcome the poor outcome of IKZF1 del and IKZF1 del/CD20+. Disclosures No relevant conflicts of interest to declare.

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Mechanisms of CRLF2 Rearrangement with IGH and P2RY8 in B-Cell Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v114.22.3243.3243 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3243-3243 ◽

Cited By ~ 1

Author(s):

David M Miller ◽

Akinori Yoda ◽

Yuka Yoda ◽

David M Weinstock

Keyword(s):

Acute Lymphoblastic Leukemia ◽

High Risk ◽

B Cell ◽

Transcriptional Control ◽

Conflicts Of Interest ◽

Single Case ◽

Lymphoblastic Leukemia ◽

Receptor Gene ◽

Great Majority ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 3243 Poster Board III-180 Introduction Precursor B-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children. While the great majority of these children achieve long-term remission, the disease exacts a high mortality in adults, and children with high-risk features. Fusion oncogenes such as BCR-ABL, MLL-AF4, E2A-PBX and TEL-AML1 are present in 60% of B-ALL cases. Cloning of these rearrangements has provided significant insight into the mechanisms involved in their formation. The gene alterations that affect the remaining 40% are poorly understood. We and others have recently identified CRLF2, a subunit of the thymic stromal lymphopoietin receptor, as a novel oncoprotein in B-ALL. Approximately 15% of adult and high-risk pediatric B-ALL that lack other characteristic gene rearrangements overexpress CRLF2, while leukemias with these rearrangements do not. In cases with CRLF2 overexpression, CRLF2 appears to be the driver of STAT activation, either alone or in combination with gain-of-function mutations in Janus Kinases (JAKs). CRLF2 overexpression results from rearrangements involving the CRLF2 locus, which is located in the pseudoautosomal regions on chromosomes X and Y. These rearrangements can either involve translocation with the immunoglobulin heavy chain (IGH) locus (t(X;14)(p22;q32) or t(Y;14)(p11;q32)) or interstitial deletion (del(X)(p22.33p22.33) or del(Y)(p11.32p11.32)). We sought to define the mechanisms of cleavage and repair that mediate these rearrangements. Methods For the translocations, we performed a series of polymerase chain reaction (PCR) assays to amplify junctions between IGHJ segments on chr.14 and the region upstream (i.e., centromeric) of CRLF2 on chr.X/Y. For the deletions, we used single nucleotide polymorphism (SNP) arrays and quantitative PCR to define the extent of deletions and then amplified junctions by PCR. Results We successfully amplified IGHJ/CRLF2 translocation junctions from six B-ALL with CRLF2 overexpression. Junctions involved chr.X/Y sequence between 8-16kb upstream of the CRLF2 translation start site. Five of 6 clustered near putative V(D)J recombinase recognition signal sequences (RSS). Additional evidence for involvement of the V(D)J recombinase was identified in all cases, including the presence of nontemplated nucleotides. In contrast, deletions resulted in juxtaposition of the full length CRLF2 coding sequence to P2RY8. A similar event involving t(X;12) that resulted in SOX5 translocation to P2RY8 has been described in a single case of splenic follicular lymphoma. P2RY8 is a member of the purine nucleotide G-protein coupled receptor gene family and is highly expressed in lymphocytes. Conclusion CRLF2 rearrangements result in overexpression through juxtaposition to alternate transcriptional control elements. While translocations appear to be mediated by aberrant V(D)J recombination, deletions likely involve an alternate sequence-dependent mechanism that targets downstream of the P2RY8 promoter. Disclosures No relevant conflicts of interest to declare.

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Frequency of IKZF1 Deletions in a Peruvian Population with B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2020-137122 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-11

Author(s):

Daniel J Enriquez ◽

Rachel J. Mitchell ◽

Krisztina Zuborne Alapi ◽

Elizabeth Cervantes ◽

Karina Cancino ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Dominant Negative ◽

White Blood Count ◽

Induction Treatment ◽

Wild Type ◽

Cell Acute Lymphoblastic Leukemia

Frequency of IKZF1 Deletions in a Peruvian Population with B-cell Acute Lymphoblastic Leukemia Background: B-cell Acute Lymphoblastic Leukemia (B-ALL) is an aggressive disease with worse outcomes in older patients, and latino ethnicity. Additionally, Latino populations are at higher risk of developing B-ALL.IKZF1is an essential lymphoid transcription factor with deletions (ΔIKZF1)implicated in treatment failure and relapses. We aimed to evaluate the frequency ofIKZF1deletions in a cohort of Peruvian patients with newly diagnosed B-ALL. Methods: We collected diagnostic bone marrow samples from 41 consecutive patients with B-ALL diagnosed between 2015-2019 at Instituto Nacional de Enfermedades Neoplasicas (INEN; Lima, Peru). Bone marrow samples were cryopreserved prior to induction treatment. DNA was extracted using High Pure PCR Template Preparation Kit (Roche) at INEN. Samples with adequate DNA were screened forΔIKZF1by multiplex endpoint PCR covering four main deletions - dominant negative Δ4-7 or the loss of function Δ2-7, Δ4-8, and Δ2-8 IKZF1 deletions at UCL Cancer Insitute (London, UK) using the primers described by Caye et. al. We analyzed outcomes byIKZF1status. Results: Forty-one cases were enrolled during the study period. Clinical characteristics are presented in Table 1. Median age was 20 years[1-63]. Fifteen∆IKZF1cases (37%) were detected (67%BCR-ABL1 negand 33%BCR-ABL1pos).Cases withΔIKZF1were older than those with wild-typeIKZF1(median age 31 vs 13 years, p=0.002). Median presenting white blood count (WBC) was 48 x109/L [R:2-218], with a higher WBC inΔIKZF1compared to wild-type (87 vs 24 x109/L, p=0.001). The most frequent deletion was ∆4-7 (sevenBCR-ABL1 negand threeBCR-ABL1 pos) additional deletions are described in table 2. All patients received intensive 'pediatric-based' treatment, 21 with BFM-2009 and 19 with the CALGB 10403 protocol. CR rates after induction were 67% and 92% for∆IKZF1and wild-type cases, respectively. Eleven (73%) of patients with∆IKZF1subjects (73%) were MRD positive by flow cytometry after induction compared to 11 (44%) among wild-type. At a median follow-up of 2 years EFS was 38% in the∆IKZF1group and 58% in the wild type group, correspond OS was 38% and 58%, respectively. Conclusion: A high frequency of IKZF1 deletions was found in a Peruvian population with B-ALL and was associated with older age and higher presenting white blood counts. Prospective studies with larger Latino population are warranted to confirm this finding. 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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Induction of NK Cell Reactivity against B-Cell Acute Lymphoblastic Leukemia by an Fc-Optimized FLT3 Antibody

Cancers ◽

10.3390/cancers11121966 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1966 ◽

Cited By ~ 1

Author(s):

Bastian J. Schmied ◽

Martina S. Lutz ◽

Fabian Riegg ◽

Latifa Zekri ◽

Jonas S. Heitmann ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Line ◽

Nk Cell ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Target Cells ◽

Dependent Manner ◽

Cell Reactivity ◽

Cell Acute Lymphoblastic Leukemia

Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism by which antitumor antibodies mediate therapeutic efficacy. At present, we evaluate an Fc-optimized (amino acid substitutions S239D/I332E) FLT3 antibody termed 4G8-SDIEM (FLYSYN) in patients with acute myeloid leukemia (NCT02789254). Here we studied the possibility to induce NK cell ADCC against B-cell acute lymphoblastic leukemia (B-ALL) by Fc-optimized FLT3 antibody treatment. Flow cytometric analysis confirmed that FLT3 is widely expressed on B-ALL cell lines and leukemic cells of B-ALL patients. FLT3 expression did not correlate with that of CD20, which is targeted by Rituximab, a therapeutic monoclonal antibody (mAb) employed in B-ALL treatment regimens. Our FLT3 mAb with enhanced affinity to the Fc receptor CD16a termed 4G8-SDIE potently induced NK cell reactivity against FLT3-transfectants, the B-ALL cell line SEM and primary leukemic cells of adult B-ALL patients in a target-antigen dependent manner as revealed by analyses of NK cell activation and degranulation. This was mirrored by potent 4G8-SDIE mediated NK cell ADCC in experiments with FLT3-transfectants, the cell line SEM and primary cells as target cells. Taken together, the findings presented in this study provide evidence that 4G8-SDIE may be a promising agent for the treatment of B-ALL, particularly in CD20-negative cases.

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An Fc-Optimized CD133 Antibody for Induction of NK Cell Reactivity against B Cell Acute Lymphoblastic Leukemia

Cancers ◽

10.3390/cancers13071632 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1632

Author(s):

Fabian Riegg ◽

Martina S. Lutz ◽

Bastian J. Schmied ◽

Jonas S. Heitmann ◽

Manon Queudeville ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Nk Cell ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Dependent Manner ◽

Cell Reactivity ◽

Cd133 Expression ◽

Cell Acute Lymphoblastic Leukemia

In recent decades, antibody-dependent cellular cytotoxicity (ADCC)-inducing monoclonal antibodies (mAbs) have revolutionized cancer immunotherapy, and Fc engineering strategies have been utilized to further improve efficacy. A promising option is to enhance the affinity of an antibody’s Fc-part to the Fc-receptor CD16 by altering the amino acid sequence. Herein, we characterized an S239D/I332E-modified CD133 mAb termed 293C3-SDIE for treatment of B cell acute lymphoblastic leukemia (B-ALL). Flow cytometric analysis revealed CD133 expression on B-ALL cell lines and leukemic cells of 50% (14 of 28) B-ALL patients. 293C3-SDIE potently induced NK cell reactivity against the B-ALL cell lines SEM and RS4;11, as well as leukemic cells of B-ALL patients in a target antigen-dependent manner, as revealed by analysis of NK cell activation, degranulation, and cytotoxicity. Of note, CD133 expression did not correlate with BCR-ABL, CD19, CD20, or CD22, which are presently used as therapeutic targets in B-ALL, which revealed CD133 as an independent target for B-ALL treatment. Increased CD133 expression was also observed in MLL-AF4-rearranged B-ALL, indicating that 293C3-SDIE may constitute a particularly suitable treatment option in this hard-to-treat subpopulation. Taken together, our results identify 293C3-SDIE as a promising therapeutic agent for the treatment of B-ALL.

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Differences In the Mechanism of CRLF2 Rearrangement In Precursor B-Cell Acute Lymphoblastic Leukemia Based on Extrasomy (21)

Blood ◽

10.1182/blood.v116.21.27.27 ◽

2010 ◽

Vol 116 (21) ◽

pp. 27-27

Author(s):

Akinori Yoda ◽

Albert Tsai ◽

Michael Lieber ◽

David M Weinstock

Keyword(s):

Down Syndrome ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Signal Sequences ◽

Cpg Dinucleotides ◽

Translocation Breakpoints ◽

Cell Acute Lymphoblastic Leukemia ◽

Activation Induced Deaminase

Abstract Abstract 27 The cytokine receptor CRLF2 is overexpressed through chromosomal rearrangement in 5–10% of adult and pediatric precursor B-cell acute lymphoblastic leukemia (B-ALL) and 60% of B-ALL in children with Down Syndrome (congenital extrasomy (21)). CRLF2 rearrangement places full-length CRLF2 under alternate transcriptional control, and can either result from an intrachromosomal CRLF2-P2RY8 deletion or from a translocation t(14;X/Y) between CRLF2-IGH. To determine whether extrasomy (21) affects the type or mechanism of CRLF2 rearrangement, we reviewed all 160 published cases of CRLF2-rearranged B-ALL. There is a striking association (Figure A) between extrasomy (21), either from Down Syndrome or somatically acquired, and CRLF2 rearrangement through CRLF2-P2RY8 deletion (p<0.001). In contrast, 63% of cases that lack extrasomy (21) harbor CRLF2-IGH translocations. CRLF2-P2RY8 deletions are known to involve off-target V(D)J recombination and occur precisely at canonical recognition signal sequences (Mullighan et al. Nat Genet 2009). To determine the mechanism(s) of CRLF2-IGH rearrangement, we analyzed all published junction sequences. Translocation breakpoints upstream of CRLF2 distribute over ∼25 kb. A single cluster of 311 bp contains 6 of 19 breakpoints, with a 36-fold “enrichment” of breakpoints in the region. We previously reported that translocation breakpoints in BCL2, BCL1, and MALT1 preferentially localize to the dinucleotide CpG, and involve a mechanism of cleavage dependent on deamination of methylated cytosine by activation induced deaminase (Tsai et al., Cell 2008). Of 19 total CRLF2 breakpoints from CRFL2-IGH rearrangements, 7 are directly at CpGs (p<0.0004; Figure B) and breakpoints are much closer to CpGs than predicted by random chance (p<10-4). In contrast, breakpoints are not significantly clustered around ▾CAC or ▾CACA (p>0.1 in all tests), the minimum sequence requirement for V(D)J recombinase recognition. Eighteen of 19 IGH-CRLF2 junctions contain N-nucleotide additions, and at least 18 of 19 of the IGH partners are compatible with standard V(D)J recombination. Thus, the CRLF2 region from IGH-CRLF2 translocations shares all the key features of other regions previously described as having CpG-type breaks: 1) strong focusing to the dinucleotide sequence CpG, with breakpoints on either side (5’ or 3’) of CpG, 2) significantly weaker or no focusing to any other dinucleotide motif or ▾CAC, 3) a propensity to cluster into 20–600 bp zones, and 4) evidence of occurrence at the pro-B/pre-B stage, including nontemplated nucleotide additions and joining to the coding ends of IGH recognition signal sequences. In conclusion, somatic or germline extrasomy(21) is closely associated with CRLF2-P2RY8 rearrangement mediated by the V(D)J recombinase. In contrast, CRLF2-IGH rearrangements involve a mechanism of cleavage at or near CpG dinucleotides also observed at BCL2, BCL1 and MALT1 in follicular, mantle cell and MALT lymphomas, respectively. Despite the differences in stage of arrested differentiation between these lymphomas and CRLF2-rearranged B-ALL, IGH translocations in each of the diseases appear to occur within pro/pre-B cells. Disclosures: No relevant conflicts of interest to declare.

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Subclonal Variation of RAG-Mediated Diversification in B Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-128660 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3955-3955

Author(s):

Carol Fries ◽

Diana G Adlowitz ◽

Philip J Rock ◽

Janice M Spence ◽

John P Spence ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Deep Sequencing ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Driver Mutations ◽

Analysis Tool ◽

Single Patient ◽

Cell Acute Lymphoblastic Leukemia

Background: Recombination-activating gene (RAG) mediates recombination of the immunoglobulin heavy chain variable gene (IGHV) in immature B lymphocytes. Aberrant targeting of RAG to non-IGH sites in B cell acute lymphoblastic leukemia (B-ALL) contributes to the development of driver mutations and clonal evolution (Papaemmanuil et al. Nat. Genet. 2014). This finding suggests that patients whose ALL involves increased RAG-mediated clonal diversification would exhibit more aggressive disease. As an initial step toward addressing this hypothesis, we asked whether the extent of RAG-mediated diversification in a patient's leukemia is consistent across all cells or variable between subclones. To assess variation in RAG-mediated subclone diversification, we interrogated rearranged IGH sequences from diagnostic B-ALL specimens to identify early subclones (SC) and to quantify RAG-derived sub-subclones (SSC) from each. We hypothesized that if RAG activity is a consistent feature of the leukemia, all SC within a single patient will have a comparable extent of SSC evolution. Methods: Amplicon-based IGHV sequencing identified the number of clonal IGH SC and their RAG-derived SSC in 22 pre-treatment adult and pediatric patients with newly-diagnosed B-ALL. Analysis was performed on peripheral blood (PB) for all patients in addition to diagnostic bone marrow (BM) for 16 of the 22 patients studied. Ultra-deep sequencing of IGH utilized 500 ng genomic DNA (representing approximately 80,000 cells/specimen) for the MiSeqDx platform, generating ~300 bp reads surrounding the VDJ junctional region. The NCBI IgBlast sequence analysis tool assigned IGH VDJ identities according to germline reference. Methods for determining subclones (SC) of shared clonal lineage involved classifying reads with a common Jh identity and 6 shared upstream Dh-Jh junctional nucleotides (termed "6N_Jx") according to defined methods (Gawad et al. Blood 2012). Further evolved sub-subclones (SCC) - as evidence of ongoing RAG activity - were defined by unique junctional sequences upstream of the common Dh-Jh junction (termed the "NDN" region). SSC were quantified according to their relative proportions within each SC family. Results: VDJ-rearranged SC families were detected for 20 of the 22 patients studied (median 2/patient; range 1-7); further analysis to assess variation in RAG-mediated diversification was limited to the 18 cases with ≥ 2 SC. In 9 of these 18, numerous evolved SSCs were identified from at least one SC in the specimen. In 4/9, there were starkly distinct levels of RAG-mediated diversity observed between intrapatient SC families, with some clonal precursors giving rise to numerous SSCs (up to 2,200 SSC per SC) while others showed minimal-to-no RAG-derived evolution (Table 1). Fifteen of 16 patients with matched BM and PB diagnostic specimens had detectable SCs. Of these, 73% (11/15) shared the dominant SC, while in 27% (4/15) the dominant SC differed between sites. However, regardless of which SC predominated, the extent of SSC diversification within each SC was preserved between the BM and the PB, with similar evolution patterns observed regardless of disease site. There was no relationship between SC read frequency and number of SSCs. Conclusions: Using deep sequencing of a single IGHV locus, wide variation in the extent of subclone diversification was observed in 4 of 20 patients with B-ALL. These findings indicate that the degree of RAG-mediated heterogeneity in B-ALL can range from minimal to extensive among distinct subclones in a single patient. The data underscore the relevance of single cell investigation of tumor characteristics to improving our understanding of the mechanisms of clonal evolution in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

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MicroRNA Mir-125b Causes Leukemia

Blood ◽

10.1182/blood.v116.21.3158.3158 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3158-3158

Author(s):

Marina Bousquet ◽

Marian Harris ◽

Beiyan Zhou ◽

Mark D. Fleming ◽

Harvey Lodish

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Macrocytic Anemia ◽

Leukemic Cells ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 3158 MicroRNA miR-125b has been shown to be involved in different kind of leukemia. Indeed, the chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b up to 90 fold. Moreover, miR-125b is also upregulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All of the mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half of them died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative disorder, suggesting an important role of miR-125b in myeloid and lymphoid lineages. Co-expression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared to control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus we showed that the overexpression of miR-125b is sufficient to induce leukemia in vivo and decrease the latency of BCR-ABL -induced leukemia. Disclosures: No relevant conflicts of interest to declare.

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STAT5 Activation Is Mandatory for IL-7-Mediated Viability and Growth of T-Cell Acute Lymphoblastic Leukemia Cells

Blood ◽

10.1182/blood.v120.21.3530.3530 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3530-3530

Author(s):

Daniel Ribeiro ◽

Ana Silva ◽

Bruno A. Cardoso ◽

Joao T. Barata

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Survival ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Leukemia Cells ◽

Dependent Manner ◽

Dismal Prognosis ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 3530 T-cell acute lymphoblastic leukemia (T-ALL) constitutes, in general, an aggressive subset of ALL, the most frequent childhood malignancy. Although risk-adjusted chemotherapeutic regimens are currently extremely effective, their efficacy is associated with significant long-term side effects and those cases that relapse have dismal prognosis. New, more specific, therapies are therefore required. To achieve this goal it is essential to have a better understanding of T-ALL biology, including the contribution of tumor microenvironmental factors for leukemia progression. Interleukin 7 (IL-7) is produced by stromal cells in the bone marrow and thymus. While IL-7 is essential for normal T-cell development, there is also considerable evidence that it can partake in leukemia expansion. Previously, we have shown that IL-7 promotes T-ALL expansion in vivo (Silva et al, Cancer Res. 2011) and leukemia cell survival and proliferation in vitro by activating PI3K/Akt/mTOR signaling pathway (Barata et al, J Exp Med. 2004), consequently leading to p27kip1 downregulation and Bcl-2 upregulation. However, it is also known that T-cell lymphomas arising spontaneously in IL-7 transgenic mice depend on STAT5 activity. Thus, we investigated whether STAT5 could also be involved in the IL-7 pro-leukemia effects in human T-ALL cells. Using an IL-7-dependent leukemia T-cell line (TAIL7), we show that IL-7 activates JAK-STAT5 pathway in a dose- and time-dependent manner, as measured by phosphorylation of JAK1, JAK3 and STAT5. We further demonstrate that IL-7 induces STAT5 DNA binding and transcriptional activity. To establish the role of STAT5, we stably transduced HPB-ALL cells with lentiviral vectors driving the expression of STAT5a shRNA or scramble control, and confirmed the efficiency of STAT5a knock down at the protein level. Flow cytometry analysis of cell size and annexin V binding, indicate that STAT5 is indispensable for IL-7-mediated T-ALL cell growth and viability. To test the potential clinical applicability of these observations, we treated TAIL7 and primary leukemia cells collected from pediatric T-ALL patients at diagnosis with pharmacological inhibitors of JAK3 (WHI-P131), STATs in general (parthenolide) and STAT5 in particular (N-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide). All three inhibitors abrogate IL-7-mediated T-ALL cell viability, growth and proliferation. At the molecular level, these observations correlate with complete inhibition of IL-7-induced modulation of p27kip1, cyclin A and cyclin D2 expression and with prevention of transferrin receptor (CD71) upregulation. Interestingly, Bcl-2 expression does not appear to be significantly affected by inhibition of STAT5 activity. This contrasts with PI3K/Akt pathway and suggests that STAT5 regulates leukemia T-cell survival, at least in part, by an alternative, Bcl-2-independent mechanism. Overall, these results extend our observations that JAK/STAT5 pathway inhibitors can eliminate cells displaying IL-7Rα gain-of-function mutations that we recently found in T-ALL (Zenatti et al, Nat Genet. 2011), and indicate that STAT5 plays a major role in mediating IL-7/IL-7R signaling effects in T-ALL cells, therefore constituting a promising target for therapeutic intervention in this malignancy. Disclosures: No relevant conflicts of interest to declare.

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