scholarly journals Targeted Activation of B Cell Autoimmunity Checkpoints in Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3716-3716
Author(s):  
Zhengshan Chen ◽  
Huimin Geng ◽  
Clifford A. Lowell ◽  
Arthur Weiss ◽  
Stephen P. Hunger ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Negative Selection ◽  
Lymphoblastic Leukemia ◽  
Cell Selection ◽  
Surface Receptors ◽  
Bcr Signaling

Abstract Background: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the B cell receptor (BCR): If B-cells fail to express a functional BCR, signaling output is too weak, resulting in "death by neglect". If the BCR binds to ubiquitous self-antigen, BCR signals are exceedingly strong. Both attenuation below minimum (non-functional BCR; death by neglect) and hyperactivation above maximum (autoreactive BCR) thresholds of signaling strength trigger negative selection and cell death. Rationale: Unlike any other types of cancer, we recently discovered that pre-B acute lymphoblastic leukemia (ALL) cells are bound by the same rules that also govern normal B cell selection. The oncogenic BCR-ABL1 tyrosine kinase mimics active pre-BCR signaling in Ph+ acute lymphoblastic leukemia which defines the ALL subgroup with the worst clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph+ ALL cells through a mechanism that is functionally equivalent to removal of self-reactive B cells. Results: ALL typically originates from pre-B cells that critically depend on survival signals emanating from a functional pre-BCR. Despite their pre-B cell origin, Ph+ ALL cells lack expression of pre-BCR signaling chains Iga and Igb, indicating lack of a functional pre-BCR. Reconstitution of Iga and Igb was indeed sufficient to induce cell death in BCR-ABL1 ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. These findings suggest that Ph+ ALL are uniquely sensitive to even incremental increases of pre-BCR signaling. Consistent with this concept, patient-derived Ph+ ALL cells express high levels of inhibitory surface receptors PECAM1, CD300A and LAIR1 that recruit and activate phosphatases SHP1 and SHIP1, which terminate pre-BCR signaling. Importantly, high expression levels of these surface receptors were strong predictors of poor outcome of patients with ALL in two clinical trials, including both pediatric and adult ALL patients. Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases SHP1 and SHIP1. Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 transformed ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of an autoreactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in Ph+ ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of SHIP1, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in human Ph+ ALL. Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

PTEN as a tumor promoter

2016 ◽  
Vol 9 (423) ◽  
pp. ec84-ec84
Author(s):  
Wei Wong
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Tumor Suppressor ◽  
B Cell ◽  
Negative Selection ◽  
Lymphoblastic Leukemia ◽  
Pharmacological Inhibition ◽  
Link Type ◽  
Factor C

PTEN is generally considered to be a tumor suppressor because it limits the activity of the PI3K-Akt pathway, which usually promotes cell survival. However, in pre-B cells transformed with BCR-ABL1 or NRASG12D, oncogenes common to acute lymphoblastic leukemia (ALL), Shojaee et al. found that deletion of Pten resulted in cell death, and mice transplanted with the transformed pre-B cells in which Pten was also deleted did not develop leukemia. Pten deletion in transformed pre-B cells resulted in increased phosphorylation of Akt, which is activated downstream of the pre-B cell receptor through the tyrosine kinase Syk. Pharmacological inhibition of Akt or Syk reduced cell death caused by Pten deletion; it also prevented the cell death of autoreactive B cells, which are eliminated through negative selection because the pre-BCR binds to self-antigen. Pten deletion did not affect the abundance of the tumor suppressor p53 or the survival of BCR-ABL1–transformed chronic myeloid leukemia (CML) cells. In contrast, Pten deletion in BCR-ABL1–transformed pre-B ALL cells triggered the phosphorylation of p53 and its accumulation, effects that required Akt activity. Overexpression of the myeloid transcription factor C/EBP-α converts cells of the B cell lineage to the myeloid lineage, and Pten deletion increased glycolysis to a greater extent in pre-B ALL cells than in myeloid-reprogrammed cells, as indicated by increased glucose consumption and lactate production and depletion of ATP. Analysis of a genetic database of human cancers indicated that PTEN deletions or point mutations were not detected in pre-B ALL patient samples, and PTEN abundance was increased in pre-B ALL patient samples compared to that in patient samples of other types of lymphomas and leukemias. PTEN knockdown reduced cell viability in four different patient-derived pre-B ALL cell lines, and pharmacological inhibition of PTEN increased AKT signaling; the phosphorylation and accumulation of p53; and glycolytic metabolism in human pre-B ALL cells. Thus, PTEN may be a potential therapeutic target for the treatment of pre-B ALL (see also Fortin et al.). S. Shojaee, L. N. Chan, M. Buchner, V. Cazzaniga, K. N. Cosgun, H. Geng, Y. H. Qiu, M. Dühren-von Minden, T. Ernst, A. Hochhaus, G. Cazzaniga, A. Melnick, S. M. Kornblau, T. G. Graeber, H. Wu, H. Jumaa, M. Müschen, PTEN opposes negative selection and enables oncogenic transformation of pre-B cells. Nat. Med. 22,379–387 (2016). [PubMed] J. Fortin, C. Bassi, T. W. Mak, PTEN enables the development of pre-B acute lymphoblastic leukemia. Nat. Med. 22, 339–340 (2016). [PubMed]

Harnessing Negative B Cell Selection to Overcome Drug-Resistance in Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 792-792
Author(s):  
Zhengshan Chen ◽  
Seyedmehdi Shojaee ◽  
Huimin Geng ◽  
Jae-Woong Lee ◽  
Maike Buchner ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
B Cell ◽  
Negative Selection ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Targets ◽  
Inhibitory Receptors ◽  
Oncogenic Signaling ◽  
Bcr Signaling ◽  
Tki Treatment

Abstract Background: B cells are selected for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: attenuation below minimum (e.g. non-functional BCR) or hyperactivation above maximum (e.g. autoreactive BCR) thresholds of signaling strength causes negative selection and cell death. About 25% of B cell acute lymphoblastic leukemia (ALL) is driven by oncogenic tyrosine kinases (e.g. BCR-ABL1 in Ph+ ALL), which mimics constitutively active pre-BCR signaling and defines the ALL subgroup with the worst clinical outcome. Currently more potent tyrosine kinase inhibitors (TKI) are developed for Ph+ ALL to suppress oncogenic signaling below a minimum threshold for survival. However Ph+ ALL cells invariably develop resistance against TKI. Here, we tested the hypothesis that targeted hyperactivation of oncogenic signaling above a maximum threshold will trigger B cell-inherent mechanisms of negative selection and selectively kill Ph+ALL cells. Results: The Ph+ ALL cells don not express a functional pre-BCR and BCR-ABL1 oncogene mimics a constitutively active pre-BCR by phosphorylating SYK, LYN, BTK and PLCg2. An incremental increase of pre-BCR downstream signaling (ITAM or SYK overexpression) was indeed sufficient to induce cell death in Ph+ ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ALL cells in this experimental setting. Ph + ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1 (CD31), CD300A and LAIR1. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials. In the COG trial (P9906; n=207) for children high-risk ALL, mRNA levels of PECAM1, CD300A and LAIR1at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p<0.0005), and negatively correlated with overall survival (OS) rate (p<0.02) or relapse free survival (RFS) rate (p<0.05). In the ECOG trial (E2993; n=215) for adults ALL, PECAM1 mRNA level negatively correlated with OS rate (p=0.0285). Genetic studies revealed that Pecam1, Cd300a and Lair1 receptors are critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases Ptpn6 (SHP1) and Inpp5d (SHIP1). Deletion of Pecam1, Cd300a or Lair1 in Ph+ ALL cells caused increased ROS levels, G0/G1cell cycle arrest, decreased colony formation capacity and cellular senescence. Phosphorylation of pre-BCR downstream molecules (SYK, LYN, BTK and PLCg2) was increased after Lair1 deletion and this hyper-signaling could not be tolerated by Ph+ ALL cells. Lair1 deletion resulted in rapid leukemia regression and prolonged survival of recipient mice in a transplant experiment. Leukemia cell death caused by Lair1-deletion could be rescued by overexpression of the active inhibitory phosphatase Ptpn6 (CD8-SHP1) or Inpp5d (CD8-SHIP1). Genetic deletion of Ptpn6 and Inpp5d caused increased pre-BCR signaling and cell death in BCR-ABL1 ALL cells but not myeloid cells (normal and BCR-ABL1-transformed), which -unlike B cells- are not subject to negative selection of auto-reactive clones. Decreasing pre-BCR signaling by SYK inhibition rescued cell death after Ptpn6- or Inpp5d- deletion. Blocking inhibitory receptors by using chimeric PECAM1, CD300A and LAIR1 receptor decoys inhibited proliferation and caused cell death in Ph+ ALL xenograft cells. More potently, a novel small molecule inhibitor of INPP5D (SHIP1) selectively killed Ph+ ALL xenograft cells through inducing hyper pre-BCR signaling, regardless of TKI resistance. We demonstrate that inhibitory phosphatase signaling represents a potential novel class of therapeutic targets for Ph+ALL. Conclusions: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified ITIM-receptors and phosphatases as members of a potential novel class of therapeutic targets. The concept of pharmacological perturbance of oncogenic signaling equilibrium in leukemia cells by inhibition (e.g. TKI-treatment) or exaggeration of signaling strength (e.g. blockade of ITIM-receptors) may lead to the discovery of multiple additional therapeutic targets and broaden our repertoire of currently available therapeutic intervention. Disclosures No relevant conflicts of interest to declare.

The Linker Protein GAS7 Negatively Regulates Pre-B Cell Differentiation and Amplifies Proliferation and Survival Signals in Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3777-3777 ◽  
Author(s):  
Jae-Woong Lee ◽  
Maike Buchner ◽  
Huimin Geng ◽  
Srividya Swaminathan ◽  
Eugene Park ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Growth Arrest ◽  
Strong Increase ◽  
Fusion Partner ◽  
B Cell Differentiation

Abstract Background: Growth arrest-specific gene 7 (Gas7) functions as an adaptor for SH2- and SH3-containing proteins, in particular in cells that undergo growth arrest. Gas7 is abundantly expressed in the brain and is involved in neuronal differentiation. Interestingly, MLL-GAS7 fusion molecules resulting from the t(11;17)(q23;p13) chromosomal translocation have been reported in treatment-related acute myeloid leukemia (AML; Megonigal et al., 2000) and in a pediatric acute lymphoblastic leukemia (ALL). While the function of MLL has been extensively studied, the role of its fusion partner GAS7 in normal hematopoiesis and leukemia has not been elucidated. Results: Studying gene expression changes during normal B cell development, we identified Gas7 as the gene with the strongest relative increase at the pre-B cell receptor checkpoint. At the transition from IL7-dependent Fraction C’ to IL7-independent small resting pre-B cells (Fraction D), GAS7 mRNA levels were upregulated by >13-fold in both human and mouse B cell progenitors. Withdrawal of IL7 cytokine signaling and Cre-mediated conditional deletion of Stat5ab recapitulated the strong increase of GAS7 expression under cell culture conditions. These finding suggest that GAS7 is part of an adaptive response of differentiating pre-B cells to attenuation of cytokine/Stat5 signaling. Consistent with this scenario, we found that Gas7-/-pre-B cells undergo accelerated differentiation, including spontaneous Ig κ light chain gene recombination and loss of Stat5-signaling. Conversely, overexpression of GAS7, reduced responsiveness of pre-B cells to normal differentiation stimuli. These findings suggest that the linker molecule GAS7 is a negative regulator of pre-B cell differentiation. Likewise, we found that tyrosine kinase inhibitor treatment of human Ph+ ALL cells resulted in a strong increased of GAS7 expression, in parallel with loss of Stat5 function. To elucidate the function of Gas7 in B cell lineage leukemia, we transformed bone marrow pre-B cells from Gas7-/- mice with BCR-ABL1. Gas7 deficient Ph+ ALL cells showed decreased proliferation with reduced S phase and increased apoptosis. In agreement with effects of Stat5 on the sensitivity of Ph+ ALL cells against tyrosine kinase inhibitors (TKIs), Gas7 deficient Ph+ ALL cells showed massively increased susceptibility to Imatinib-induced apoptosis. In addition, absence of Gas7 caused loss of self-renewal capacity and failure to form colonies in methylcellulose assay. Co-immunoprecipitation experiments with flag tagged GAS7 in patient-derived Ph+ALL cells revealed that GAS7 physically interacts with STAT5 and retains STAT5-Y694 in an active conformation.Thereby, GAS7 can propagate even weak Stat5 activity and maintain residual cytokine or BCR-ABL1 oncogenic signaling in normal and malignant pre-B cells. Conclusions: Here show that GAS7 functions as an important positive regulator of Stat5 downstream of cytokine receptors in normal pre-B cells and downstream of BCR-ABL1 and other oncogenes in leukemia. Owing to the GAS7-dependent reinforcement of Stat5-dependent survival and proliferation signaling, normal and leukemic pre-B cells can survive periods of reduced cytokine/oncogene signaling. These findings suggest that the interaction interface between GAS7 and Stat5 represents a potential target for small molecule scaffolds and peptides. Disclosures No relevant conflicts of interest to declare.

scholarly journals Co-Expression of SYK and ZAP70 Subverts Negative B-Cell Selection and Enables Oncogenic Signaling in Multiple B-Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 295-295
Author(s):  
Teresa Sadras ◽  
Mickaël Martin ◽  
Lauren Kim-Sing ◽  
Jevon Cutler ◽  
Gal Lenz ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Cell Selection ◽  
B Cell Malignancies ◽  
Close Proximity ◽  
Mantle Cell ◽  
Bcr Signaling

B-cells are under intense selective pressure to eliminate autoreactive or premalignant clones. B-cell receptor (BCR) signals are required for survival, however, BCR-signaling exceeding maximum thresholds often reflects signaling from an autoreactive BCR or a transforming oncogene and triggers negative selection and cell death. The tyrosine kinase SYK initiates BCR-downstream signaling in B-cells while its close relative ZAP70 is almost exclusively expressed in T-cells. Interestingly, the segregation of SYK to B-cells and ZAP70 to T-cells is less confined in malignant lymphopoiesis suggesting that the balance of these related kinases may alter signaling output in disease and contribute to development of leukemia. As previously shown in B-cell chronic lymphocytic leukemia (B-CLL), we identified aberrant ZAP70 expression as a frequent feature in multiple other B-cell malignancies that depend on survival signals from a functional (pre-) BCR (E2A-PBX1+ pre-B ALL, and mantle cell lymphoma) or harbor oncogenic mimics of the BCR (BCR-ABL1+ B-ALL). Studying SYK and ZAP70 expression by single-cell Western blot, co-expression of the two tyrosine kinases was extremely rare in normal B- and T-cell populations. In contrast, &gt;50% of tumor B-cells in mantle cell lymphoma, pre-B ALL and CLL co-expressed SYK and ZAP70. Despite their structural similarities, genetic deletion and engineered reconstitution of SYK and ZAP70 in human B-cell lymphoma cells revealed striking functional differences. Proximity-dependent biotin identification (BioID) analyses identified that SYK, but not ZAP70, engaged the PI3K pathway via interaction with CD19. Consistent with this, reconstitution with SYK and SYK-ZAP70 but not ZAP70 alone promoted survival and proliferation. Detailed analysis of BCR-mediated cascades in lymphoma cells expressing SYK, ZAP70 or SYK-ZAP70 established that ZAP70 is only weakly efficient at propagating BCR-mediated calcium and downstream pathway activation in B-cells. Strikingly, co-expression of ZAP70 with SYK resulted in re-wired BCR-signaling of intermediate strength: compared to cells expressing only SYK, SYK-ZAP70 co-expressing cells had markedly reduced activation of the BLNK-BTK-PLCγ pathway, further reflected in BCR-induced Ca2+ signaling with delayed onset, lower amplitude but longer duration. In this way, we speculated that SYK and ZAP70 may be present within close proximity at the apex of BCR-initiated interactions, and hence compete for downstream substrates resulting in a re-wiring of classic signaling programs propagated normally by SYK. To explore this, we utilized proximity ligation assays (PLA) to monitor the proximity of SYK and ZAP70 in resting or BCR-stimulated B-cells, and found that SYK and ZAP70 co-exist within close proximity consistent with the view that varying levels of these kinases may alter B-cell signaling output. Functional experiments further showed that phosphomimetic activation of SYK, but not ZAP70, induced hyperactivation of PI3K-signaling and acute BTK-mediated cell death in pre-B ALL cells. In line with altered BCR-signaling strength and quality in SYK and ZAP70 co-expressing cells, over-expression of Zap70 in pre-B ALL cells rescued auto-immune checkpoint activation induced by hyper-activation of BCR-associated signaling. To study functional consequences of SYK-ZAP70 co-expression during normal B-cell development, we generated a novel knock in Zap-70+/Mb1-Cre+mouse model, to induce conditional expression of Zap70 in the B cell compartment from the proB stage. Consistent with compromised central tolerance checkpoints, Syk-Zap70 co-expressing pro/pre-B and immature B-cells had reduced spontaneous apoptosis rates and gave rise to autoantibody production against multiple self-antigens. Importantly, our findings highlight a previously unrecognized role for ZAP70 in oncogenic BCR-signaling and we conclude that the co-expression of ZAP70 mitigates the ability of SYK, downstream of an autoreactive BCR or a transforming oncogene, to trigger negative B-cell selection and cell death (Figure 1). Disclosures Weinstock: Celgene: Research Funding. Meffre:AbbVie: Consultancy, Other: Grant.

Rac2 GTPase Activation Is Necessary for Development of p190-BCR-ABL-Induced B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3790-3790
Author(s):  
Abel Sanchez-Aguilera ◽  
Ami tava Sengupta ◽  
Joseph P Mastin ◽  
Kyung H Chang ◽  
David A Williams ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Rho Gtpases ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Cell Acute Lymphoblastic Leukemia

Abstract The fusion gene BCR-ABL, resulting from t(9;22) reciprocal chromosomal translocations, encodes a constitutively active tyrosine kinase. Two different isoforms of BCR-ABL, p190 and p210, are associated to two completely different diseases. In the tyrosine kinase inhibitor (TKI) era, while p210-BCR-ABL-induced CML is highly responsive to TKI, p190-BCR-ABL still induces a poor prognosis B-cell acute lymphoblastic leukemia (B-ALL). The only difference between these two forms of BCR-ABL is the existence of a DH/Cdc24/PH domain in p210-BCR-ABL, which acts as a guanine nucleotide exchange factor (GEF) able to activate Rho GTPases. Rac is a subfamily of Rho GTPases with regulatory activity on hematopoietic stem cell and progenitor (HSC/P) functions. We have previously shown that Rac2 and further the combination of Rac1 and Rac2 mediate downstream signals in p210 BCR-ABL-induced myeloproliferation (Thomas EK, et al., Cancer Cell, 2007). Interestingly, despite the absence of a GEF domain in p190-BCR-ABL, Rac is activated, suggesting the activation of other GEF(s). Here we have analyzed whether Vav and Rac family members are involved in p190-BCR-ABL-induced B-ALL. We have used a combination of in vitro (Ba/F3 pro-B cells transduced with p190 or p210 BCR-ABL) and in vivo (murine transduction-transplantation model of p190 BCR-ABL-induced B-ALL) approaches. In Ba/F3 cells, both p190 BCR-ABL and p210 BCR-ABL activated Rac and the Rac effector p21 activated kinase (PAK), and their proliferation and survival appeared severely decreased in response to the Rac activation inhibitor NSC23766. Stat3, Stat5 and Jnk, but not ERK, p38 or NF-kB, were constitutively hyperactivated in p190 BCRABL-expressing Ba/F3 cells and primary murine B-ALL cells. Intracellular flow cytometry analysis demonstrated that Stat5 was specifically activated in the pro/pre-B leukemic cell population, compared to normal B cells. In the murine model of B-ALL, loss of Rac2, but not Rac3, prolonged survival and impaired leukemia development. Like in Ba/F3 cells, primary B-CFU and outgrowth in Witte-Whitlock assays of leukemic primary cells from mice was severely decreased by the addition of NSC23766 to the culture. Although Vav was activated by both p190- and p210-BCR-ABL, since NSC23766 does not block the activation by Vav1, we hypothesized that other GEFs were involved. Indeed, the loss of Vav1 or even combined loss of Vav1 and Vav2 did not impair BCR-ABL-mediated lymphoid leukemogenesis in vivo. Vav3, another member in the Vav family which uses a different mechanism of activation of Rac GTPases was a likely candidate. In fact, loss of Vav3 alone was able to significantly prolong the survival and attenuate development of p190 BCR-ABL-driven B-ALL. In conclusion, the results of this study indicate that Rac activation is necessary for the development of B-ALL induced by p190-BCR-ABL in vitro and in vivo, and validate a new signaling pathway as a therapeutic target for BCR-ABL-induced B-ALL.

Identification of Genes Abnormally Expressed in Human MLL-AF4 Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4314-4314
Author(s):  
Shuangli Mi ◽  
Miao Sun ◽  
Zejuan Li ◽  
Roger T. Luo ◽  
Nimanthi Jayathilaka ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Candidate Genes ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Cells ◽  
Chromosome 11

Abstract The t(4;11)(q21;q23) translocation is a hallmark of infant acute lymphoblastic leukemia (ALL), which results in the fusion of the MLL gene on chromosome 11 and the AF4 gene on chromosome 4. MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukemia and is associated with a poor prognosis. To identify leukemia-related genes, we used the SAGE technique to compare gene expression profiles between two MLL-AF4 patient samples and one normal sample (CD19+ progenitor B cells; 216,464 tags in total). We identified 61 candidate genes that appear to be abnormally expressed in the leukemia samples (29 up- and 32 down-regulated). Remarkably, we found that many candidate genes appear to play important role in the development of B cells. In addition, many candidate genes can bind with and/or regulate other candidates in the candidate gene list. For example, SYK, BTK and BLNK can bind directly and regulate each other. SYK can also bind directly with TNFRSF1B. In addition, EBF may positively regulate BLK, while BLK can bind directly with BTK. All six of these genes are significantly down-regulated in MLL-AF4 leukemia samples. BTK, SYK and BLK are tyrosine kinases. BTK (B-cell progenitor tyrosine kinase) is a key regulator in B-lymphocyte differentiation and activation. BLK (B-lymphocyte-specific tyrosine kinase) is expressed only in B lymphocytes, and controls pre-B cell development. SYK (spleen tyrosine kinase) is widely expressed in hematopoietic cells, which can phosphorylate BLNK (B-cell linker protein). BLNK represents a central linker protein that bridges the B-cell receptor-associated kinases and may regulate B-cell function and development. EBF (early B-cell factor) is a tissue-specific and differentiation stage-specific DNA-binding protein, and mice lacking Ebf are unable develop B lymphoid cells. TNFRSF1B is strongly expressed on stimulated T and B lymphocytes. Moreover, previous studies indicate that BTK, BLK, SYK, BLNK and TNFRSF1B can positively regulate apoptosis, while BTK can also positively regulate differentiation. Thus, their down-regulation may inhibit apoptosis and differentiation, and thereby contribute to leukemogenesis. In contrast, GNA12, a transforming oncogene which can enhance proliferation and transformation and can bind directly with BTK, is significantly up-regulated in MLL-AF4 leukemia cells. Its up-regulation may also be important to leukemogenesis. Taken together, the deregulation of the important candidate genes may contribute to leukemogenesis through inhibiting apoptosis and differentiation while promoting proliferation of hematopoietic cells. We have validated the expression patterns of the candidate genes with real-time quantitative RT-PCR and are studying the functions and pathways of the validated candidate genes using RNAi and retrovirus transduction over-expression methods. In addition, we will also establish knock-in or knock-out mouse models for the most promising functional candidate genes to see the effect on the development of leukemia. Our studies will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute lymphoblastic leukemia, which may lead to more effective therapy for these leukemias.

Efficacy of PRT060318, a Novel Highly Specific SYK Inhibitor, in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3532-3532
Author(s):  
Stefan KÖhrer ◽  
Greg Coffey ◽  
Ekaterina Kim ◽  
Nathalie Y Rosin ◽  
Uma Sinha ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Phenotype ◽  
Bcr Signaling ◽  
Syk Inhibitor ◽  
Induction Of Apoptosis

Abstract Abstract 3532 B cell acute lymphoblastic leukemia (B-ALL) arises by transformation of progenitor (pre-B) cells. Cure rates in adults remain low and treatment is complicated by microenvironment-mediated resistance to cytotoxic drugs, indicating an urgent need for development of new, more targeted treatment approaches. Spleen tyrosine kinase (Syk), a B cell receptor (BCR)-associated tyrosine kinase, recently was identified as a novel therapeutic target in mature B cell malignancies, such as chronic lymphocytic leukemia (CLL). Besides its role in BCR signaling in mature B cells, Syk also plays an important role in maintenance and expansion of immature B cells. Syk-deficient mice display a severe defect of B lymphopoiesis, with a block at the pro-B to pre-B transition, consistent with a key role for Syk in pre-BCR signaling. We therefore hypothesized that pre-B ALL cells which express pre-BCRs might be more sensitive to Syk inhibition than other ALL subtypes, which either do not have functional pre-BCRs (pro-B ALL) or display modifying genetic lesions (Ph+/BCR-ABL+ B-ALL). PRT060318, a highly specific, ATP-competitive, small molecular inhibitor of Syk, has preclinical activity in CLL and DLBCL models (Hoellenriegel J et al., Leukemia 26:1576–83, 2012). Syk specificity was confirmed by cellular and non-cellular kinase inhibition assays. We performed metabolic assays using the tetrazolium dye XTT to screen B-ALL cell lines (RS4;11, REH, TOM-1, Nalm-20, Nalm-21, Nalm-6, 697, Kasumi-2, KOPN-8, RCH-ACV, SMS-SB) for responses to PRT060318. ALL cells were incubated with increasing concentrations of PRT060318 (100 nM – 10 μM) for 72 hours. Based on the XTT assays, we determined half maximal inhibitory concentration (IC50), and separated B-ALL cells into responsive (IC50 < 4.5 μM) and non-responsive (IC50 > 4.5 μM) groups. Interestingly, the responsive group consisted only of pre-B ALL cells (CD10+, TdT+, cytoIgμ+), whereas the resistant group comprised only ALL cell lines with a pro-B cell phenotype (CD10+/−, TdT+, cytoIgμ-). All BCR-ABL positive cell lines tested exhibit the pro-B cell phenotype and, similar to their BCR-ABL negative counterparts, were resistant to PRT060318. The Figure depicts XTT assay results for two PRT060318-sensitive pre-B (grey lines) and two resistant pro-B (black lines) B-ALL cell lines. Western Blot analysis demonstrated SYK protein expression in all B-ALL cell lines, excluding lack of target protein expression as possible cause for different response rates between pre-B and pro-B ALL cells. Cell proliferation assays revealed a significant, dose-dependent inhibition of pre-B ALL cell proliferation by PRT060318. Concordantly, dose depended S phase reduction was detected in all PRT060318-sensitive cell lines. In apoptosis assays, in which sensitive pre-B ALL lines were incubated with increasing concentrations of PRT060318 (up to 5 μM) we did not find any significant induction of apoptosis, suggesting anti-proliferative, rather than cytotoxic effects as a main mechanism of action of PRT060318. Preliminary data of primary ALL patient samples, cultured with KUSA-H1 marrow stroma cells in the presence or absence of PRT060318 demonstrate induction of apoptosis in 3 out of 10 samples, other assays are ongoing. In conclusion, we provide evidence that the Syk inhibitor PRT060318 thwarts pre-B ALL cell proliferation, providing a first rationale for clinical testing of PRT060318 in selected patients with B cell ALL. Disclosures: Coffey: Portola Pharmaceuticals Inc.: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment. Pandey:Portola Pharmaceuticals Inc.: Employment.

scholarly journals NUP214-ABL1 fusion defines a rare subtype of B-cell precursor acute lymphoblastic leukemia that could benefit from tyrosine kinase inhibitors

Haematologica ◽  
2015 ◽  
Vol 101 (4) ◽  
pp. e133-e134 ◽  
Author(s):  
Nicolas Duployez ◽  
Guillaume Grzych ◽  
Benoît Ducourneau ◽  
Martin Alarcon Fuentes ◽  
Nathalie Grardel ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor

B-Cell Acute Lymphoblastic Leukemia Arising in Patients with a Preexisting Diagnosis of Multiple Myeloma Is a Novel Cancer with High Incidence of TP53 Mutations

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Monique Chavez ◽  
Erica Barnell ◽  
Malachi Griffith ◽  
Zachary Skidmore ◽  
Obi Griffith ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Mechanism Of Action ◽  
Lymphoblastic Leukemia ◽  
Treatment Options ◽  
Hematopoietic Stem ◽  
Tp53 Mutations ◽  
Cell Acute Lymphoblastic Leukemia

Multiple Myeloma (MM) is a malignancy of plasma cells that affects over 30,000 Americans every year. Despite advances in the treatment of the disease, approximately 12,000 American patients will still die of MM in 2019. One of the mainstays of treatment for MM is the immunomodulatory and antiangiogenic drug lenalidomide; which is used in induction therapy, maintenance therapy and treatment of relapsed disease. Although not fully elucidated, lenalidomide's mechanism of action in MM involves the drug binding to Cerebelon (CBN) and leads to the subsequent degradation of the Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors (TF). These TFs play important regulatory roles in lymphocyte development. Despite lenalidomide's importance in MM treatment, several groups have reported that MM patients treated with lenalidomide rarely go on to develop B-cell acute lymphoblastic leukemia (B-ALL). The genetics and clonal relationship between the MM and subsequent B-ALL have not been previously defined. Importantly, it is not clear if the MM and B-ALL arise from the same founding clone that has been under selective pressure during lenalidomide treatment. As deletions in IKZF1 are common in B-ALL, one could hypothesize that lenalidomide's mechanism of action mimics this alteration and contributes to leukemogenesis. We sequenced the tumors from a cohort of seven patients with MM treated with lenalidomide who later developed B-ALL. These data did not show any mutational overlap between the MM and ALL samples-the tumors arose from different founding clones in each case. However, several genes were recurrently mutated in the B-ALL samples across the seven patients. These genes included TP53, ZFP36L2, KIR3DL2, RNASE-L, and TERT. Strikingly, five of the seven patients had a TP53 mutations in the B-ALL sample that was not present in the matched MM sample. The frequency of TP53 mutations in our cohort was much higher than that reported in adult de novo B-ALL patients which can range between 4.1-6.4% (Hernández-Rivas et al. 2017 and Foa et al. 2013). Utilizing CRISPR-Cas9 gene editing, we disrupted the Zfp36l2 or Actb in murine hematopoietic stem cells (HSCs) of mice with or without loss of Trp53. We performed our first transplantation experiment in which the cohorts of mice have loss of Trp53 alone, loss of Zfp36l2 alone, loss of both Trp53 and Zfp36l2, or a control knockout (KO) of Actb. To characterize the disruption of Zfp36l2 alone and in combination with Trp53 we analyzed the hematopoietic stem and progenitor cell compartments in the bone marrow of the above transplanted mice. In mice with a loss of Zfp36l2 there is a decrease in Lin- Sca-1+ c-Kit+ (LSK), short term-HSC (ST-HSC), and multipotent progenitors (MPP). This decrease was not observed in the mice with a loss of both Trp53 and Zfp36l2, where instead we noted an increase in monocyte progenitors (MP), granulocytes-macrophage progenitors (GMP), and common myeloid progenitors (CMP) cells. In this Trp53 Zfp36l2 double loss model we also noted a decrease in B220+ B-cells that was not seen in the Zfp36l2 alone. In this cohort of Trp53 Zfp36l2 loss, we characterized B-cell development through hardy fraction flow cytometry, and identified a decrease in fractions A and B/C (pre-pro and pro-B-cells, respectively) as compared to Zfp36l2 or Actb alone. As lenalidomide does not bind to Cbn in mice, we used the human B-ALL NALM6 cell line to test if treatment with lenalidomide will lead to a selective growth advantage of cells with the same genes knocked out versus wild-type control cells grown in the same culture. We hypothesize that lenalidomide treatment selectively enriched for pre-existing mutated cell clones that evolved into the B-ALL. Preliminary data in NALM6 cells with a loss of TP53 demonstrate a slight increase in cell number at day 7 compared to a RELA control. These experiments will be repeated with concurrent ZFP36L2 and TP53 mutations as well as ZFP36L2 alone. Treatment-related disease is a key consideration when deciding between different treatment options, and this project aims to understand the relationship between MM treatment and B-ALL occurrence. It may be possible to identify MM patients who are at-risk for B-ALL. For example, MM patients who harbor low-level TP53 mutations prior to lenalidomide treatment could be offered alternative treatment options. Disclosures Barnell: Geneoscopy Inc: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Wartman:Novartis: Consultancy; Incyte: Consultancy.

scholarly journals Src homology 2 domain–containing inositol-5-phosphatase and CCAAT enhancer-binding protein β are targeted by miR-155 in B cells of Eμ-MiR-155 transgenic mice

Blood ◽  
2009 ◽  
Vol 114 (7) ◽  
pp. 1374-1382 ◽  
Author(s):  
Stefan Costinean ◽  
Sukhinder K. Sandhu ◽  
Irene M. Pedersen ◽  
Esmerina Tili ◽  
Rossana Trotta ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
High Grade ◽  
Src Homology 2 ◽  
Enhancer Binding Protein ◽  
Src Homology ◽  
Src Homology 2 Domain

AbstractWe showed that Eμ-MiR-155 transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre–B-cell proliferation, have variable clinical presentation, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain–containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein β (C/EBPβ), 2 important regulators of the interleukin-6 signaling pathway, are direct targets of MiR-155 and become gradually more down-regulated in the leukemic than in the preleukemic mice. We hypothesize that miR-155, by down-modulating Ship and C/EBPβ, initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma.

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