pre-B Cell Receptor Research Articles

Involvement of Blnk and Foxo1 in tumor suppression in BCR‑ABL1‑transformed pro‑B cells.

Oncogenic Bcr-Abl kinase mimics pre-B cell receptor (pre-BCR) survival signals in BCR-ABL1-positive B-cell acute lymphoblastic leukemia (BCR-ABL1+ B-ALL), driving B-cell progenitor malignant transformation; thus, defining a particularly unfavorable prognosis for patients. During B-cell development, pre-BCR differentiation signaling components terminate proliferative expansion and promote B-cell maturation. To study whether pre-BCR differentiation signaling components regulate the initiation and development of BCR-ABL1+ B-ALL, the tumor suppression mechanism of differentiation-related signaling molecules in BCR-ABL1-transformed pro-B cells were analyzed. The results demonstrated that Bcr-Abl kinase activated the PI3K/Akt pathway, promoting cell growth, and upregulated Aid expression, increasing genomic instability in pro-B cells. These findings suggest that Bcr-Abl kinase mediates pro-B cell malignant transformation. Furthermore, the present data revealed that BCR-ABL1 oncogenic stress triggered enhanced expression of B-cell differentiation components B-cell linker (Blnk) and forkhead box protein O1 (Foxo1) in BCR-ABL1 transformed pro-B cells. Using the CRISPR/Cas9-mediated Blnk or Foxo1 knockout BCR-ABL1-transformed pro-B cells, it was identified that, in BCR-ABL1-transformed pro-B cells, Blnk and Foxo1 reduced Bcr-Abl kinase activity to induce cell cycle arrest and decrease genomic instability. In addition, Blnk suppressed the PI3K/Akt pathway to reduce Foxo1 phosphorylation and heighten the Foxo1 activity, indicating that, in BCR-ABL1-transformed pro-B cells, Foxo1 participated in the regulation of Bcr-Abl kinase by Blnk. The present data highlighted the antitumor mechanisms of Blnk and Foxo1 in the regulation of Bcr-Abl kinase, and thus, may offer an alternative therapeutic strategy to Bcr-Abl kinase regulation in BCR-ABL1+ B-ALL.

Efficacy of high-dose corticosteroid-based treatment for chronic lymphocytic leukemia patients with p53 abnormalities in the era of B-cell receptor inhibitors

PurposeHigh-dose methylprednisolone (HDMP) with or without anti-CD20 antibody treatment in the pre B-cell receptor inhibitor (BCRi) era was used as potential salvage therapy for relapsed/refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (r/r CLL/SLL) patients bearing the 17p deletion. Patients and methodsOutcomes were compared in retrospect between r/r patients treated with HDMP (n = 20), ibrutinib (n = 39) and idelalisib with rituximab (n = 14). ResultsHigher overall response rates were found in those patients undergoing BCRi therapy compared to HDMP (79.2% vs. 0%; p < 0.0001), along with longer median progression-free survival (not reached vs. 24.1 months; p < 0.01). Nevertheless, there were no differences in the overall survival (HDMP 35.87 months vs. not reached; p = 0.58). ConclusionHDMP treatment was significantly inferior in terms of response rate and progression-free survival in r/r CLL/SLL patients with the 17p deletion, and may only be used whenever novel compounds are unavailable.

Early B cell protein λ5 affects skeletal homeostasis

Abstract There are strong indications that B lineage cells play a key role in bone homeostasis. The preB cell receptor is composed of an immunoglobulin mu H chain, VpreB and immunoglobulin λ5. Young λ5 deficient mice have reduced numbers of preB cells and mature B cells, moreover; their bone appears to be normal. With time, they achieve normal numbers of mature B cells while exhibiting major bone loss. To test whether expression of λ5 is playing a critical role maintaining bone homeostasis, we evaluated bone in: mice lacking immunoglobulin JH (JH −/− λ5+/+). These mice lack both preB and mature B cells, but maintain expression of λ5 in progenitor B cells. Mice deficient in both JH and λ5 (JH −/− λ5−/−) have no preB or mature B cells, and also no λ5. Using both μCT and histologic evaluation, we compared femur and skull at six months of age in these three mutant strains to normal controls. JH −/− λ5+/+ femur and skull were similar to WT. Both JH +/+λ5−/− and JH −/− λ5−/− mice showed significant decrease in femoral trabecular bone and increased loss of skull bone. Irradiated WT mice transplanted with JH −/− λ5−/− bone marrow cells developed defective femoral and cranial bones. In contrast, JH −/−λ5−/− mice that received WT bone marrow cells recovered bone architecture. In vitro culture of bone progenitors from JH +/+λ5−/− and JH −/− λ5−/− mice showed decreased ability of bone formation. These data underscore the importance of immunoglobulin λ5 in skeletal development.

Oncogene-Independent Adaptation of Pre-B Cell Receptor Signaling Confers Drug Resistance and Signaling Plasticity in Ph-like ALL

Oncogene-Independent Adaptation of Pre-B Cell Receptor Signaling Confers Drug Resistance and Signaling Plasticity in Ph-like ALL

Genetic Approaches for Definitive Diagnosis of Agammaglobulinemia in Consanguineous Families.

Autosomal recessive agammaglobulinemia (ARA) is a primary immunodeficiency characterized by absent peripheral B cells, severe hypogammaglobulinemia, and absent BTK gene mutations. In ARA, mutations occur in genes encoding the pre-B cell receptor (pre-BCR) or downstream signaling proteins. In this work, we used candidate gene and whole-exome sequencing to investigate the molecular basis of ARA in 6 patients from 4 consanguineous North-African families. Sanger sequencing of candidate genes encoding the pre-BCR components (ΙGΗΜ, CD79A, CD79B, IGLL1, and VPREB1) was initially performed and determined the genetic defect in five patients. Two novel mutations in IGHM (p.Val378Alafs*1 and p.Ile184Serfs*21) were identified in three patients from two unrelated kindred and a novel nonsense mutation was identified in CD79A (p.Trp66*) in two siblings from a third kindred. Whole-exome sequencing (WES) was performed on the sixth patient who harbored a homozygous stop mutation at position 407 in the RAG2 gene (p.Glu407*). We concluded that conventional gene sequencing, especially when multiple genes are involved in the defect as is the case in ARA, is costly and time-consuming, resulting in delayed diagnosis that contributes to increased morbidity and mortality. In addition, it fails to identify the involvement of novel and unsuspected gene defects when the phenotype of the patients is atypical. WES has the potential to provide a rapid and more accurate genetic diagnosis in ARA, which is crucial for the treatment of the patients.

Disruption of the preB Cell Receptor Complex Leads to Decreased Bone Mass.

In the bone marrow, preB cells are found adjacent to the bone endosteum where bone synthesizing osteoblast and bone resorbing osteoclasts reside. Although there is evidence of interactions between preB and bone cells, the factors that contribute to such interactions are poorly understood. A critical checkpoint for preB cell development assesses the integrity of the nascent immunoglobulin μ heavy chain (HC) by testing whether it can participate in the formation of a preB cell receptor (preBCR), composed of the μ HC and surrogate light chain (LC). In this work, we tested whether loss of preBCR components can affect bone synthesis. A panel of gene targeted mice with sequential blocks in preBCR formation or function [surrogate light chain component lambda 5 deleted (λ5−/−), transmembrane domain of μHC deleted (IgM-mem−/−), and CD19 preBCR co-receptor deleted (CD19−/−)] were evaluated for effects on postnatal bone synthesis. Postnatal bone mass was analyzed in 6 month old mice using μ-CT, histomorphometry and double calcein labeling. Both cortical and trabecular bone mass were significantly decreased in the femurs of the λ5 and IgM-mem deficient mice. Histomorphometric analysis showed a decrease in the numbers of osteoblasts and osteoclasts in all three mutant strains. Double calcein labeling revealed a significant decrease in dynamic synthesis and mineralization of bone in λ5−/− mice. Our data strongly suggest that interference with preBCR formation or function affects bone homeostasis independent of the presence or absence of mature B cells, and that components of the preBCR play important, and potentially distinct, roles in regulating adult bone mass.

O-GlcNAcylation of c-Myc dynamically regulates pre-B cell proliferation

Abstract O-GlcNAc modification regulates the activity of hundreds of nucleocytoplasmic proteins involved in a wide variety of cellular processes such as gene expression, signaling and cell growth. However, the mechanism underlying the regulation of B cell development and function by O-GlcNAcylation is largely unknown. Here, we demonstrate that changes in cellular O-GlcNAc levels significantly affect the growth of large pre-B cells transiently expressing pre-B cell receptors on their surface membrane, which are known to rapidly proliferate for the expansion of functional clones that express successfully rearranged heavy chains during early B cell development. Our study first show that overall O-GlcNAc levels in these proliferative pre-B cells are highly induced compared to pro-B cells, depending on pre-B cell receptor expression. Mechanistically, inhibition of O-GlcNAcylation in pre-B cells markedly decreases c-Myc expression, which results in downregulation of cell proliferation through the blocking of E- and A-type cyclin expressions. In addition, restriction of glucose uptake in pre-B cells also reduces cellular O-GlcNAc levels as well as c-Myc expression and subsequently decreases cell proliferation. Furthermore, the population of large pre-B cells in the bone marrow is significantly reduced by the administration of O-GlcNAc inhibitor to mice. Thus, our study strongly suggests that O-GlcNAcylation dependent expression of c-Myc is a new component in regulation of pre-B cell proliferation and also a potential therapeutic target for the treatment of pre-B cell acute lymphoblastic leukemia.

Tungsten Blocks Murine B Lymphocyte Differentiation and Proliferation Through Downregulation of IL-7 Receptor/Pax5 Signaling.

Tungsten is an emerging environmental toxicant associated with several pediatric leukemia clusters, although a causal association has not been established. Our previous work demonstrated that tungsten exposure resulted in an accumulation of pre-B cells in the bone marrow, the same cell type that accumulates in pediatric acute lymphoblastic leukemia (ALL). To better understand the relevant molecular mechanisms, we performed RNA-sequencing on flow sorted pre-B cells from control and tungsten-exposed mice. Tungsten decreased the expression of multiple genes critical for B cell development, including members of the interleukin-7 receptor (IL-7R) and pre-B cell receptor signaling pathways, such as Jak1, Stat5a, Pax5, Syk, and Ikzf3. These results were confirmed in an in vitro model of B cell differentiation, where tungsten arrested differentiation at the pro-B cell stage and inhibited proliferation. These changes were associated with decreased expression of multiple genes in the IL-7R signaling pathway and decreased percentage of IL-7R, phosphorylated STAT5 double-positive cells. Supplementation with IL-7 or overexpression of Pax5, the transcription factor downstream of IL-7R, rescued the tungsten-induced differentiation block. Together, these data support the hypothesis that IL-7R/Pax5 signaling axis is critical to tungsten-mediated effects on pre-B cell development. Importantly, many of these molecules are modulated in ALL.

Precursor B-cell development in bone marrow of Good syndrome patients

Good syndrome is an immunodeficiency presenting with thymoma, hypogammaglobulinemia and almost absent B cells. To investigate the origin of the B-cell lymphopenia in these patients, we studied B cell differentiation in the bone marrow of Good syndrome patients. We found very low numbers of precursor B cells in bone marrow of Good syndrome patients and a differentiation arrest after the pro-B-cell stage; this is different from other agammaglobulinemia patients with a defect in pre B-cell receptor signaling.

Significant In Vivo Sensitivity to Aurora Kinase Inhibition in TCF3-Hlf rearranged Acute Lymphoblastic Leukemia

Significant In Vivo Sensitivity to Aurora Kinase Inhibition in TCF3-Hlf rearranged Acute Lymphoblastic Leukemia

CD79a Is Associated with Central Nervous System Infiltration of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

▪Despite the advances in the treatment of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), infiltration of the central nervous system (CNS) remains a clinical challenge. Certain cytogenetic subtypes such as E2A-PBX1-and BCR-ABL-positive BCP-ALL confer a higher risk for CNS involvement initially and for CNS relapse. Novel strategies to predict CNS and to eradicate leukemic cells from the CNS are subjects of ongoing research.In order to identify targets with diagnostic and therapeutic relevance, comparative RNA-sequencing was performed with patient derived xenograft (PDX) blasts from 5 E2A-PBX1-positive patients, recovered from the bone marrow (BM) and from the CNS of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Differential gene expression analysis revealed the upregulation of various genes of the pre-B cell receptor complex, particularly the signaling component CD79a (Igα) in blasts recovered from the CNS as compared to blasts from the BM. We then investigated the impact of CD79a on CNS infiltration in vivo and in patients. CD79a was downregulated by short-hairpin RNA (shRNA) mediated knockdown in the E2A-PBX1 positive cell line 697. Proliferation rates of 697-shCD79a cells and control-transfected 697 cells (697-shCtrl) in vitro were similar. Furthermore, NSG mice injected with 697-shCD79a cells showed comparable survival times, as well as similar blast infiltration in spleen and BM as animals injected with 697-shCtrl cells. However, downregulation of CD79a led to a significantly lower number of CNS-positive mice (4/15, 26%) as compared to control animals (7/10, 70%) (p=0.0486, Figure A). This indicates that CD79a is not critically involved in proliferation and peripheral engraftment, but in CNS infiltration of E2A-PBX1 positive 697 cells in vivo.To test if CD79a also affects CNS involvement in BCR-ABL-positive leukemia, a murine/murine transplantation model was used. B-cells isolated from CD79a-knockout (CD79a-KO) or wildtype mice (CD79a-Ctrl) were stably transfected with a BCR-ABL fusion gene and cultured independent of cytokines, thereby inducing malignant transformation. Both cell lines were subsequently injected into recipient NSG mice (n=8/group) and leukemic development was followed. The experiment was terminated when all control mice had developed leukemic symptoms and mice were analyzed for leukemic engraftment. A further CD79a-KO group was included for survival analysis. Median spleen volume as a surrogate of leukemic infiltration was significantly lower in mice injected with CD79a-KO as compared to CD79a-Ctrl cells (0.35 cm³ vs. 0.06 cm³; p=0.0001). Median blast percentages in spleens and BM were also markedly reduced (75.3% vs. 5.8%; p=0.0001 and 61.0% vs. 4.5%; p=0.0001, respectively). Importantly, none of the animals in the CD79a-KO group showed blasts in the CNS as assessed by histology whereas blasts were present in all of the animals in the CD79a-Ctrl group. Finally and most importantly, NSG-mice injected with CD79a-KO cells showed a highly significant prolongation in median survival as compared to mice with CD79a-Ctrl cells (29 days vs. 95 days; p=0.0001, Figure B). Altogether, these data suggest that in a model of BCR-ABL-positive leukemia, absence of CD79a impacts the engraftment of blasts in vivo, in the CNS and other leukemic niches.To further validate our findings in patient material, we measured CD79a protein expression in PDX cells from an E2A-PBX1- and a BCR-ABL-positive patient serially transplanted into NSG mice for three passages. For both entities and in all passages, CNS blasts showed a higher CD79a expression than blasts isolated from the bone marrow. In order to assess if CD79a can be used as a marker to predict CNS involvement in patients, CD79a mRNA levels were measured in a selected cohort of 98 pediatric BCP-ALL patients, which contained 26 CNS-positive patients matched to 72 CNS-negative patients. CNS-positive patients showed significantly higher mRNA levels of CD79a than CNS-negative patients (p=0.0225, unpaired t-test, Figure C) suggesting that CD79a may be of value as a potential diagnostic marker for initial CNS involvement in BCP-ALL.Our results indicate a role of CD79a in proliferation and CNS infiltration of BCP-ALL blasts in experimental settings and patients. We intend to prospectively evaluate CD79a as a prognostic marker, which may also be a therapeutic target in CNS-positive BCP-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Divergent Evolutionary Trajectories of Erk- and Stat5-Activating Lesions in Acute Lymphoblastic Leukemia

Background and significance: Activation of STAT5- and ERK-signaling are segregated to early and later stages of normal B cell development, respectively. While pro-B cells depend on activation of Stat5 downstream of cytokine receptors, pre-B and immature B cells depend on Erk-activation downstream of pre-B cell receptor (pre-BCR). Since Stat5- (cytokine receptor) and Erk- (pre-BCR) signaling antagonize each other, we studied evolutionary trajectories of Stat5- and Erk-activating lesions in B-cell lineage ALL. We tested the hypothesis that an initial activating mutation in one of the two pathways represents a commitment step that makes the tumor clone non-permissive to activating lesions in the other pathway.Results: Studying B-lineage ALL (n=578), we found that STAT5-activating (e.g. BCR-ABL1, JAK2, cytokine receptors) and ERK-activating (NRAS, KRAS, PTPN11, NF1) lesions were mutually exclusive, with only 9 cases (1.6%) carrying lesions in both pathways. Even in the few remaining cases, low allele frequencies of one of the two lesions suggests that they do not co-occur in the same cells. Reverse phase protein array measurements revealed that phosphorylation of ERK and its upstream kinase MEK were inversely correlated with STAT5-phosphorylation (MDACC, 1983-2007; P<0.001). These findings prompted us to study mechanisms of biochemical cross-inhibition between Erk and Stat5 pathways. In support of this notion, we found that inducible NRASG12D activated Erk at the expense of Stat5 phosphorylation. This was due to stabilization and increased activation of the Stat5-phosphatase Ptpn6 (SHP1). Inducible ablation of Ptpn6 elevated phospho-Stat5 levels, while genetic inactivation of Stat5 strongly increased Erk activity. Studying a matched patient-derived pre-B ALL sample at diagnosis and at relapse (acquired KRASG12V mutation) revealed activation of ERK in association with increased BCL6 and decreased Stat5 levels in the KRASG12V relapsed ALL sample. With engagement of BCL6 and the Stat5-inhibitory phosphatase Ptpn6 downstream of Erk signaling, these findings suggest that occupancy of either Erk or Stat5-pathway represents a commitment step that renders cells non-permissive to the respective alternative pathway. To test this hypothesis, we induced B cell transformation with either Erk or Stat5-pathway oncogenes and then subsequently transduced with either empty vectors or vectors carrying the alternative oncogene. Compared to empty vector controls, Erk- and Stat5-driven B-ALL cells did not tolerate transformation with the alternative oncogene. To test the antagonistic relationship between Stat5- and Erk-activating lesions in B-ALL, we transformed Stat5fl/fl B-ALL cells with NRASG12D for Erk-activation and Erk2fl/fl B-ALL with BCR-ABL1 for activation of Stat5. While Erk-driven B-ALL clones were non-permissive to transduction with Stat5-activating oncogenes (BCR-ABL1), Cre-mediated inducible ablation of Stat5 increased competitive fitness and colony formation ability. Likewise, Stat5-driven B-ALL clones were non-permissive to transduction with Erk-activating oncogenes (NRASG12D). However, Cre-mediated inducible ablation of Erk2 increased competitive fitness and colony formation ability.Conclusion: These results support a model of clonal evolution that selects malignant B-ALL clones based on increased polarization in favor of one single dominant pathway of oncogenic signal transduction with concurrent silencing of alternative pathways. This concept suggests that malignant transformation is not only associated with progressively increased signaling strength of the central oncogenic driver but also concomitant silencing of cytokine and growth factor receptors that normal cells would respond to. DisclosuresWhite:BMS: Research Funding; Novartis: Honoraria, Research Funding.

Ras-Driven B-Cell Transformation Targets Developmental Rewiring of Cytokine to Pre-B Cell Receptor Signaling

Ras-pathway lesions are oncogenic drivers in ~45% of B-cell acute lymphoblastic leukemia (B-ALL) cases. Activating mutations of NRAS and KRAS are oncogenic drivers in B-ALL while the BRAFV600E mutation occurs in almost all cases of B-cell hairy cell leukemia. Less frequent lesions resulting in increased ERK-signaling in B-cell malignancies include activating mutations of RAF1, MAP2K1 and the PTPN11 phosphatase as well as deleterious mutations of the Ras-GTPase activator NF1. Interestingly, increased immunoglobulin light chain gene expression was observed in B-ALL cases with RAS-pathway lesions (COG P9906), reflecting engagement of pre-B cell receptor (pre-BCR) downstream signaling. Here we tested the hypothesis that oncogenic RAS-signaling in B-ALL mimics pre-BCR-induced developmental rewiring of signal transduction at the pro-B to pre-B cell transition and identified PTPN6 and BCL6 as therapeutic targets in RAS-driven B-ALL.During early B-cell development, pro-B cells transition from cytokine- to pre-B cell receptor (pre-BCR)-dependent survival and proliferation signals. Inducible activation of immunoglobulin (Ig) µ heavy chain (µHC) expression induced developmental progression and surface expression of Ig κ light chains. Notably, inducible activation of oncogenic NRASG12D had the same effect and resulted in increased surface expression of Ig κ light chains. Furthermore, studying genetic models for this transition revealed that both pre-BCR signaling and RAS-oncogenes suppressed cytokine receptor/STAT5-signaling and induced massive de novo expression of the proto-oncogene and transcriptional repressor BCL6. Our genetic studies revealed that the SH2-domain containing protein tyrosine phosphatase PTPN6 was activated by oncogenic RAS-signaling and essential for the switch from STAT5 to BCL6-activation. Given that oncogenic RAS activated PTPN6, we tested the role of PTPN6 in RAS-driven leukemogenesis. To this end, ablation of Ptpn6 in NRASG12D-driven B-ALL resulted in depletion of cells from cell culture in competitive-growth assays and reduced the number of colonies formed in semi-solid methylcellulose. Collectively, these findings suggest that PTPN6 represents a potential therapeutic intervention point in RAS-driven B-ALL.In addition to PTPN6, we investigated the role of BCL6 in RAS-driven B-ALL. Aberrant activation of oncogenic RAS results in oncogene-induced senescence (OIS) characterized by induction of ARF/p53 and irreversible cell cycle arrest in the G1 phase. For oncogenic Ras-signaling, BCL6 was required to oppose ERK-mediated activation of p21, p27 and p53 checkpoint molecules in B-ALL. Here we tested the hypothesis that BCL6 bypasses the RAS-mediated OIS program to facilitate transformation. To this end, increases in number of colonies formed in semi-solid methylcellulose were observed upon inducible activation of Bcl6 in NRASG12D B-ALL cells. Furthermore, loss of Bcl6 function in NRASG12D B-ALL cells resulted in depletion of cells from cell culture in competitive growth assays and reduced colony forming ability. Importantly, expression of NRASG12D in Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In striking contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in vivo. Furthermore, pharmacological inhibition of BCL6 restored sensitivity to chemotherapy in patient-derived KRASG12V B-ALL cells.In conclusion, we identified oncogenic RAS-signaling as functional mimics of pre-BCR signaling. Oncogenic RAS induced expression of BCL6 at the expense of cytokine receptor/STAT5-signaling. Our genetic studies identified PTPN6 as a critical effector molecule of the switch from cytokine receptor to pre-BCR signaling. Importantly, we identified PTPN6 and BCL6 as potential therapeutic intervention points in RAS-driven B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pre-BCR Surrogate Light Chain Components VPREB1 and IGLL1 Function As Pre-BCR-Independent Tumor Suppressors in Acute Lymphoblastic Leukemia

Background:IGLL1 and VPREB1 encode for the l5 and VpreB components, respectively, of the surrogate light chain (SLC) of the pre-B cell receptor (pre-BCR). During early B-cell development, immunoglobulin (Ig) heavy chains pairs with SLCs to form the pre-BCR, a central signaling unit that drives proliferation and survival. Accordingly, germline mutations of IGLL1 (Minegishi J Exp Med 1998) and VPREB1 (Conley Immunol Rev 2005) are associated with profound B-cell defects and agammaglobulinemia in humans. However, somatic deletions of VPREB1 gene are frequent lesions in B-ALL and occur in >10% of B-ALL cases (Mangum et al., Leukemia 2014; Geng et al., Cancer Cell 2015), the significance of which is not clear. Since VPREB1 deletions are typically present at the time of diagnosis and are rarely acquired as secondary lesions at the time of relapse (Kuster Blood 2011), we hypothesized that loss of VPREB1 represents an early and essential event in leukemogenesis.Experimental Approach and Results: For genetic gain and loss of function analysis of VPREB1 and IGLL1, we established leukemia models based on pre-B cells from Igll1-/- mice (Kitamura Cell 1992), and Vpreb1-Igll1 double-transgenic mice (Van Loo Immunity 2007). Loss of Igll1 completely abrogated SLC expression on the surface of pre-B cells. In contrast, VpreB1-Igll1 double-transgenic pre-B cells expressed constitutively higher surface levels of SLC as part of their pre-BCR as evidenced by flow cytometry. Compared to wildtype controls, Igll1-/- pre-B cells lacking the ability to express a functional SLCs were more readily transformed by BCR-ABL1 oncogene. However, pre-B cells of VpreB1-Igll1 transgenic mice, were not permissive to BCR-ABL1 mediated transformation.In agreement with these results, VpreB1-Igll1 double-transgenic pre-B cells were resistant transformation by BCR-ABL1 in vivo. BCR-ABL1-transgenic mice with enforced expression Vpreb1-Igll1 remained disease-free for more than nine months, whereas the vast majority of BCR-ABL1-transgenic mice downregulated pre-BCR surface expression and developed lethal B-ALL within 90 days of birth (n=34, P<0.0001). Compared to wildtype pre-B cells, double-transgenic expression of VpreB-Igll1 interfered with oncogenic BCR-ABL1 tyrosine kinase signaling and suppressed phosphorylation of Btk, Syk and Src kinases resulting in cell cycle arrest and reduced colony formation ability. To test whether SLC tumor suppressive function depends on pre-BCR activity, we studied BCR-ABL1-mediated transformation of VpreB1-Igll1 double-transgenic pre-B cells on a Rag1-deficient background. Rag1-/- pro-B cells lack the ability to rearrange Ig V, D and J-gene segments and cannot express a functional Ig mHC, the central structural element of the pre-BCR. Surprisingly, Rag1-/-VpreB1-Igll1 double-transgenic pro-B cells were also resistant to BCR-ABL1-mediated transformation. These findings provide genetic evidence that Vpreb1 and Igll1 exert tumor suppressive effect on B cells regardless of functional pre-BCR expression.Conclusion: The VpreB and Igll1 surrogate light chain components of the pre-BCR act as a tumor suppressors in pre-B ALL cells. Interestingly, the tumor suppressor function of both VpreB and Igll1 is independent from their SLC-role in pre-BCR signaling: Vpreb1- and Iggl1-mediated tumor suppression was effective, regardless of pre-BCR function. These findings provide genetic evidence that VpreB and Igll1 have pre-BCR- and SLC-independent functions that prevent malignant transformation and limit proliferation of normal pre-B cells. Our findings support the hypothesis that VPREB1 deletion represents an early event during clonal evolution towards pre-B ALL, facilitating subsequent steps of leukemic transformation. DisclosuresNo relevant conflicts of interest to declare.

The Role of the Pre-B Cell Receptor in B Cell Development, Repertoire Selection, and Tolerance.

Around four decades ago, it had been observed that there were cell lines as well as cells in the fetal liver that expressed antibody μ heavy (μH) chains in the apparent absence of bona fide light chains. It was thus possible that these cells expressed another molecule(s), that assembled with μH chains. The ensuing studies led to the discovery of the pre-B cell receptor (pre-BCR), which is assembled from Ig μH and surrogate light (SL) chains, together with the signaling molecules Igα and β. It is expressed on a fraction of pro-B (pre-BI) cells and most large pre-B(II) cells, and has been implicated in IgH chain allelic exclusion and down-regulation of the recombination machinery, assessment of the expressed μH chains and shaping the IgH repertoire, transition from the pro-B to pre-B stage, pre-B cell expansion, and cessation.

Pathogenesis and recurrence factors in E2A-PBX1 fusion gene positive children with acute lymphoblastic leukemia

The translocation of(1; 19)(q23; p13)leading to E2A-PBX1 gene fusion, is one of the most common translocations in pediatric B-ALL.Fusion of the E2A-PBX1 gene forms a specific transcription factor activating the pre-B cell receptor, allowing the pre-B cells to self-renew.This process acts as a leukemia initiation factor.Simultaneously, it can combine a second mutation to activate the genetic damage activating kinase driven signaling pathway.Thereby clonality of precursor B cells proliferated abnormally, which will lead to the occurrence of leukemia.This article summarizes the secondary mutations and kinases associated with E2A-PBX1 positive recurrent patients, and provides help for targeted therapy in recurrent patients. Key words: E2A-PBX1; Pathogenesis; Recurrence factors

Maintenance and pharmacologic targeting of ROR1 protein levels via UHRF1 in t(1;19) pre-B-ALL.

Expression of the transmembrane pseudokinase ROR1 is required for survival of t(1;19)-pre-B-cell acute lymphoblastic leukemia (t(1;19) pre-B-ALL), chronic lymphocytic leukemia, and many solid tumors. However, targeting ROR1 with small-molecules has been challenging due to the absence of ROR1 kinase activity. To identify genes that regulate ROR1 expression and may, therefore, serve as surrogate drug targets, we employed an siRNA screening approach and determined that the epigenetic regulator and E3 ubiquitin ligase, UHRF1, is required for t(1;19) pre-B-ALL cell viability in a ROR1-dependent manner. Upon UHRF1 silencing, ROR1 protein is reduced without altering ROR1 mRNA, and ectopically expressed UHRF1 is sufficient to increase ROR1 levels. Additionally, proteasome inhibition rescues loss of ROR1 protein after UHRF1 silencing, suggesting a role for the proteasome in the UHRF1-ROR1 axis. Finally, we show that ROR1-positive cells are twice as sensitive to the UHRF1-targeting drug, naphthazarin, and undergo increased apoptosis compared to ROR1-negative cells. Naphthazarin elicits reduced expression of UHRF1 and ROR1, and combination of naphthazarin with inhibitors of pre-B cell receptor signaling results in further reduction of cell survival compared with either inhibitor alone. Therefore, our work reveals a mechanism by which UHRF1 stabilizes ROR1, suggesting a potential targeting strategy to inhibit ROR1 in t(1;19) pre-B-ALL and other malignancies.

Single-cell developmental classification of B cell precursor acute lymphoblastic leukemia at diagnosis reveals predictors of relapse.

Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'developmentally dependent predictor of relapse' (DDPR), significantly improves currently established risk stratification methods. DDPR features exist at diagnosis and persist at relapse. By leveraging a data-driven approach, we demonstrate the predictive value of single-cell 'omics' for patient stratification in a translational setting and provide a framework for its application to human cancer.

In the Absence of Central pre-B Cell Receptor Selection, Peripheral Selection Attempts to Optimize the Antibody Repertoire by Enriching for CDR-H3 Y101.

Sequential developmental checkpoints are used to “optimize” the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires μHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies.

Age-associatedBcells expanded in autoimmune mice are memory cells sharing H-CDR3-selected repertoires.

Age-associated Bcells (ABCs) represent a distinct cell population expressing low levels of CD21 (CD21-/low ). The Ig repertoire expressed by ABCs in aged mice is diverse and exhibits signs of somatic hypermutation (SHM). A CD21-/low B-cell population is expanded in autoimmune diseases, e.g. systemic lupus erythematosus, as well as in lupus-prone NZB/W mice and in mice lacking a pre-Bcell receptor (SLC-/- ). However, the nature of the CD21-/low Bcells (hereafter ABCs) in autoimmunity is not well understood. Here we show that in young SLC-/- mice, the vast majority of the ABCs express memory B-cell (MBC) markers in contrast to wild-type controls. A similar population is present in lupus-prone MRL mice before and at disease onset. In SLC-/- mice, a majority of the ABCs are IgM+ , their VH genes have undergone SHM, show clonal diversification and clonal restriction at the H-CDR3 level. ABC hybridomas, established from SLC-/- mice, secrete typical lupus autoantibodies, e.g. anti-Smith antigen, and some of those that bind to DNA comprise a H-CDR3 that is identical to previously described IgM anti-DNA antibodies from lupus-prone mice. Together, these results reveal that ABCs in autoimmune mice are comprised of autoreactive MBCs expressing highly restricted H-CDR3 repertoires.