pre-B Cell Receptor Signaling Research Articles

DNA Damage Signals Repress SYK in Early B Cells to Limit Leukemic Transformation

Early B cell development requires a balance of antagonistic cell programs that drive cellular proliferation as well as maturation. In large pre-B cells, pre-B cell receptor (pre-BCR) signaling activates the ETS family transcription factor, PU.1, and the tyrosine kinase, SYK, which together promote cell proliferation. Subsequently, cells undergo cell cycle arrest and transition to the small pre-B cell stage where immunoglobulin light chain (Igl) gene rearrangement is initiated. Igl gene assembly proceeds through DNA double-stranded breaks (DSBs) generated by the RAG endonuclease. These RAG DSBs induce cellular programs that inhibit pre-BCR signaling through activation of the transcription factor NFkB2. NFkB2 upregulates expression of the SPIC/BCLAF1 transcriptional repressor complex, which displaces PU.1 from the Syk promoter, resulting in reduction of SYK and enforcement of cell cycle arrest. Pediatric pre-B cell leukemia can be driven by errant pre-BCR signaling and increased SYK. We propose that B cell development relies on signals from RAG DSBs to counter pre-BCR signaling and attenuate SYK expression, thereby enforcing a pre-B cell checkpoint that promotes Igl rearrangement and suppresses leukemic transformation. To examine coordination between pre-BCR and the RAG DSB signals in vivo, we developed a novel reporter mouse with a knock-in allele at the endogenous Syk locus encoding an N-terminal fusion of mCherry to SYK (SykmCh). Heterozygous SykmCh/+ mice express equivalent SYK and mCherry-SYK proteins and have normal SYK activity as evidenced by phosphorylation of BLNK, a downstream target of the SYK kinase. To determine if SYK levels are modulated by RAG DSB signals, we generatedRag1-/-:SykmCh/+:IgH:Bcl2 and Art-/-:SykmCh/+:IgH:Bcl2 mice. Rag1-/- mice cannot make DSBs whereas Art-/- mice cannot repair DSBs, due to lack of the DNA damage response protein, Artemis. IgH transgene promotes development to pre-B cell stage and Bcl2 supports survival to permit assessment of DNA damage responses. Flow cytometry revealed lower mCherry-SYK levels in Art-/- small pre-B cells compared to Rag1-/- small pre-B cells, which supports that SYK is suppressed in vivo by RAG DSBs. We next assessed SYK levels during normal B cell development. mCherryhigh and mCherrylow small pre-B cells from SykmCh/+mice were sorted and demonstrated an inverse correlation between SYK and DSB signals. Specifically, relative to mCherryhigh small pre-B cells, mCherrylow small pre-B cells have lower SYK and increased NFkB2 activation, indicating concurrent repressed pre-BCR signals and active RAG DSB signals. Notably, mCherryhigh small pre-B cells have expression of DSB-induced genes Spic, Pim2, and Cd40. Together, these results demonstrate that RAG DSB signals are initiated in SYK-high small pre-B cells and then subsequently promote transition to SYK-low small pre-B cells. To investigate consequences of altered SYK regulation, we generated p53f/f:Bclaf1f/f:Mb1-cre mice, which have selective loss of p53 and Bclaf1 in early B cells. We previously showed that Bclaf1flox/flox:Mb1-cre mice have increased numbers of large pre-B cells with higher Syk expression. However, there were no abnormalities in immature B cell numbers, suggesting additional mechanisms, such as activation of p53 by RAG DSBs, may complement BCLAF1 to enforce B cell development. Indeed, we find that all p53flox/flox:Bclaf1flox/flox:Mb1-cre mice die between 9-12 months of age with malignant expansion of B cells in both spleen and bone marrow. In contrast, no p53flox/flox:Mb1-cre, Bclaf1flox/flox:Mb1-cre, or Bclaf1flox/flox:p53flox/flox mice died or developed abnormal B cells in this time period. The atypical B cells in the p53:Bclaf1 double-deficient mice have altered B220 expression, normal expression of IgM and absence of CD43, consistent with an aberrant immature B cell phenotype. These results demonstrate that RAG DSB signals function to preserve pre-B cell checkpoint and maintain normal B cell maturation. Collectively, these studies establish a novel system for quantitating DNA damage signaling in vivo and for assessing dynamic changes in SYK during B cell maturation. Further, our findings demonstrate a critical function of RAG DSB signals in regulation of early B cell development.

Preclinical Evidence for the Efficacy of CD79b Immunotherapy in B Cell Precursor Acute Lymphoblastic Leukemia

Patients with B cell precursor acute lymphoblastic leukemia (BCP-ALL) have a favorable prognosis, yet current treatment protocols are based on intensive cytotoxic chemotherapy and therapy options are limited when patients relapse. Novel immunotherapy approaches are therefore needed. We recently reported that the preB cell receptor signaling unit CD79a (known as Igα) is crucial for BCP-ALL engraftment in vivo, particularly in the central nervous system (CNS) employing BCR-ABL + and E2A-PBX1 + patient derived xenograft (PDX) models (Lenk et al., Communications Biology, 2021). CD79a forms a heterodimer with CD79b (known as Igß), which is also expressed on the surface of mature B cells as part of the B cell antigen receptor. Accordingly, the CD79b antibody drug conjugate (ADC) Polatuzumab Vedotin (PolVed) has shown therapeutic efficacy in the treatment of refractory/relapsed (r/r) diffuse large B cell lymphoma. Moreover, CD79a/CD79b may also be present on the cell surface at the pro/pre B cell stage. We therefore hypothesize that CD79b can serve as a therapeutic target in BCP-ALL.First, to substantiate that CD79b is important for BCP-ALL engraftment in vivo, a murine/murine transplantation model was applied. B cell precursors were isolated from mice harboring CD79b with a non-functional signaling domain (CD79b-ITAM-KO) or wildtype mice, malignantly transformed with a BCR-ABL fusion construct and transplanted into NSG-mice. Whereas control cells caused overt leukemia in all animals within 25 days, no animal injected with CD79b-ITAM-KO cells had developed leukemia by the time of sacrifice at 162 days (P<0.001).To investigate the frequency of surface (s)CD79b expression in BCP-ALL patients, we measured sCD79b levels via flow cytometry in diagnostic samples of pediatric BCP-ALL patients with different cytogenetic backgrounds. We detected sCD79b-positivity (defined as ≥10% sCD79b + BCP-ALL cells) in 23/94 patients including BCR-ABL +, E2A-PBX1 +, MLL-rearranged (MLLr), TEL-AML1 + and B-other BCP-ALL patients indicating a population of sCD79b + patients within different cytogenetic BCP-ALL subgroups (Figure 1a).To validate CD79b as a therapeutic target, we applied an unconjugated monoclonal CD79b antibody (CD79b-mAB) to PDX mice bearing either pediatric BCR-ABL + or E2A-PBX1 + PDX samples with high sCD79b expression (49.6% sCD79b + cells and 37.7% sCD79b + cells, respectively). Treatment was initiated one day after BCP-ALL injection, modelling a minimal residual disease (MRD) situation. Therapy with CD79b-mAB (1mg/kg) resulted in a mild reduction of leukemia burden in the spleen (SP) and bone marrow (BM) of PDX mice and a significant reduction of CNS-involvement in both PDX-models as compared to control treated mice (P<0.05 and P<0.01, respectively). To test the hypothesis that treatment of BCP-ALL with a CD79b-ADC outperforms CD79b-mAB, we applied PolVed (1mg/kg) in NSG mice bearing the same E2A-PBX1 + and BCR-ABL + PDX cells. PolVed therapy resulted in a significant reduction of BCP-ALL engraftment in SP, BM and CNS (P<0.01, respectively) and a significant survival prolongation compared to control treated mice in both models (P<0.01, respectively). Of note, 4/5 PolVed-treated E2A-PBX1-PDX animals were free of leukemia by the time of sacrifice (236 days) (Figure 1B).To test the efficacy of PolVed on different BCP-ALL subgroups, we conducted a preclinical phase II-like PDX study, using sCD79b high and sCD79b low PDX samples (defined by sCD79b-expression above or below the median) (5E2A-PBX1 +, 3 BCR-ABL +, 2 MLLr, 1 E2A-HLF + and 1 ETV6-NTRK3 +). Two NSG mice per patient were injected with PDX cells, randomly assigned into treatment groups and PolVed therapy was initiated when 1% PDX-cells were detected in the peripheral blood, modelling an overt leukemia situation. sCD79b low PDX mice did not respond to PolVed treatment (Figure 1c), but we detected a response to therapy and a significant survival prolongation in 5/6 sCD79b high PDX samples irrespective of the cytogenetic background (P<0.05) (Figure 1d).Taken together, our data indicate that a subgroup of BCP-ALL patients is sCD79 + positive and may respond to PolVed treatment. Therefore, we suggest CD79b as a novel therapeutic target in BCP-ALL and propose PolVed as a potential therapeutic agent in r/r disease.*LL and DW contributed equally to this work [Display omitted] DisclosuresLenk: OSE Immunotherapeutics: Research Funding. Richter: HTG Molecular Diagnostics, Inc.: Current Employment, Research Funding. Schrappe: SHIRE: Other: research support; JazzPharma: Honoraria, Other: research support; Servier: Honoraria, Other: research support; SigmaTau: Other: research support; Novartis: Honoraria; JazzPharma: Honoraria; Novartis: Honoraria, Other: research support; Servier: Honoraria; Amgen: Other: research support. Cario: Novartis: Other: Lecture Fee. Brüggemann: Incyte: Other: Advisory Board; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Schewe: Jazz Pharmaceuticals: Other: Advisory Board; SOBI: Other: Advisory Board; Bayer: Other: Advisory Board; OSE Immunotherapeutics: Research Funding.

A Spic/BCLAF1 Complex Suppresses Pre-B Cell Receptor Signaling in Response to RAG-Mediated DNA Breaks

B cell development is carefully orchestrated to generate a diverse antibody repertoire while preventing malignant transformation. At early developmental stages, the pre-B cell receptor (pre-BCR) is an essential regulator of B cell maturation, coordinating both clonal expansion and immunoglobulin light chain gene (Igl) assembly. Igl gene rearrangement proceeds through V(D)J recombination, which requires the generation of DNA double-stranded breaks (DSBs) and the subsequent joining of two distant DNA segments. The DSBs necessary for Igl gene rearrangements are generated by the RAG1 and RAG2 proteins, which form the RAG endonuclease. Once RAG DSBs are generated, cells must be prevented from re-entering cell cycle where DSBs can be aberrantly repaired as deleterious translocations. Thus, temporal segregation of pre-BCR driven proliferation and Igl gene assembly is essential for normal maturation and prevention of errors that could lead to leukemic transformation. The pre-BCR signals through the SYK tyrosine kinase to both drive proliferation and initiate Igl gene rearrangement. Unopposed activation of the pre-BCR, particularly increased SYK activity, blocks B cell development and supports leukemic transformation. Furthermore, pre-B leukemic blasts depend on SYK for proliferation and survival. Tight regulation of pre-BCR signaling is essential for enforcing normal maturation and suppressing leukemic transformation.We previously showed that pre-BCR signaling is inhibited by signals from RAG DSBs. Specifically, RAG DSBs trigger ATM-dependent induction of SPIC, an ETS family transcriptional repressor with significant homology to PU.1 and SPIB. SPIC inhibits expression of the SYK tyrosine kinase resulting in suppression of pre-BCR signaling, inhibition of pre-B cell proliferation, and limitation of additional RAG DSB generation. In order to determine the mechanism of SPIC-mediated gene regulation and to determine the role of SPIC in early B cell development, we compared SPIC and PU.1 binding throughout the genome in early B cells. Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) identified shared binding sites for SPIC and PU.1 throughout the genome in pre-B cells suggesting that both transcription factors regulate a similar cohort of genes. Interestingly, binding of SPIC to the Syk promoter and the Igl(kappa) enhancer results in reduction of PU.1 binding at these sites and suppression of transcription of the respective genes. These results demonstrate that SPIC antagonizes PU.1 function to repress pre-BCR signaling in early B cells.SPIC shares a DNA binding domain motif with the other ETS-family transcription factors, PU.1 and SPIB, but has a unique transactivation domain, which could recruit transcriptional repressors to gene regulatory regions. To determine the unique binding partners of SPIC and PU.1, we generated pre-B cells expressing tetracycline-inducible FLAG-SPIC and FLAG-PU.1. The tagged proteins were purified from nuclei using tandem affinity purification. Mass spectrometry of the precipitated samples identified BCLAF1 (Bcl2 associated transcription factor 1) as a unique SPIC interactor. In contrast, IRF4 binds to PU.1 but not to SPIC. In pre-B cells, BCLAF1 binds to regulatory regions of a similar set of genes as SPIC. Consistent with this, BCLAF1 binds to the Syk promoter only in cells with RAG DSBs, which correlates with binding of SPIC and loss of PU.1 at this site. RNAi-mediated knockdown of BCLAF1 results in increased Syk mRNA expression. However, loss of BCLAF1 does not affect SPIC occupancy at the Syk promoter. Thus, SPIC binds DNA but only represses transcription if BCLAF1 is also recruited. Collectively, our findings reveal that, in response to RAG DSBs, SPIC and BCLAF1 cooperate to antagonize PU.1 function and regulate expression of a broad genetic program that modulates pre-BCR signaling to enforce normal B cell maturation. DisclosuresNo relevant conflicts of interest to declare.

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.

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.

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.

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.

Redundant JAK, SRC and PI3 Kinase Signaling Pathways Regulate Cell Survival in Human Ph-like ALL Cell Lines and Primary Cells

▪Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk disease with a frequency of about 20% in patients of all ages. Ph-like ALL can be characterized in different groups based on their chromosomal rearrangements. The most common rearrangements involve CRLF2, ABL1, and EPOR and lead to activation of JAK kinase dependent activation of STAT5, PI3 kinase and other signaling pathways. We have previously shown that Ph-like ALL is a high-risk disease with a poor prognosis in older adults. The focus of this study is to further understand the biology of Ph-like ALL and how the cells develop drug resistance by modulating their signaling pathways to escape apoptosis.Results: We and others have identified activated STAT5 and AKT signaling in Ph-like ALL cell lines and patient samples. Interestingly, inhibition of the JAK-STAT signaling pathway using JAK-kinase inhibitors abrogated short term STA5 signaling as well as AKT signaling, but Ph-like ALL cells showed only a mild apoptotic effect or arrest in cell proliferation in vitro. Furthermore, even though xenotransplantation models of Ph-like ALL show a decrease in leukemia burden when treated with ruxolitinib, a JAK1/2 inhibitor, all mice ultimately succumb to the disease, demonstrating that Ph-like ALL does not demonstrate oncogene addiction. Based on these results we hypothesize that Ph-like ALL cells can adapt to JAK kinase inhibition by activating other signaling pathways. To test this hypothesis, we performed a time course experiment in Ph-like ALL cell lines and detected AKT reactivation shortly after JAK-kinase inhibition. In addition, we detected activation of SYK and a reduction in the PI3K negative regulator PTPN6. These observations lead to the hypothesis that Ph-like ALL may have expression and activation of the pre-B cell receptor (pre-BCR). Indeed, gene expression data from the clinical trial P9906 demonstrate that Ph-like ALL cases with CRLF2-rearrangment and JAK2 mutations express high mRNA levels of pre-B cell receptor signaling molecules, including BLNK, IGGL1, VpreB3, and ZAP70. We next hypothesized that pre-BCR mediated activation of PI3 kinase rescues Ph-like ALL cells from JAK inhibition. To study PI3K signaling we first treated Ph-like ALL cell lines and xenografts with idelalisib or INCB-50465, both selective PI3Kδ inhibitors. PI3K inhibition had only a minor effect on cell viability and again AKT was reactivated 48h later. Importantly, combining both JAK-kinase inhibition with PI3K inhibition, prevented AKT reactivation and had strong synergistic effect on cell viability and cell proliferation in vitro. Xenotransplantation models of Ph-like ALL with ruxolitinib and INCB-50465 showed a significantly lower leukemia burden as compared to the single agent treatments but disease eradication was not achieved. Pre-BCR signaling can also activate SRC family kinases and we hypothesized that CRLF2-rearanged Ph-like ALL might be sensitive to SRC inhibition as well. Strikingly, combining JAK2, PI3K, and SRC inhibition further reduced the cell viability by one log scale compared to the JAK2/PI3K inhibitor treated samples and less than 0,2% viable cells were detected, while other ALL subtypes were not affected. In vivo validation of treatment strategy is pending.Clinical relevance: While children with ALL currently have a 5-year survival rate of 90%, adults, especially those over the age of 40, have a very poor clinical outcome with survival rates of about 40%. Adults often do not tolerate high doses of chemotherapy regimens and are therefore in clinical need of alternative treatment regimens. We therefore combined JAK1/2 and PI3K inhibitory treatments with dasatinib, a frequently-used tyrosine kinase inhibitor that is well tolerated by older patients. Strikingly, the combinatory treatment had a strong effect on cell viability and represents so far, the first study using specific signaling inhibitors combined in Ph-like ALL.Conclusion: These findings identify that Ph-like ALL are not oncogene addicted like Ph+ ALL. While BCR-ABL1 activates multiple signaling pathways that can be inhibited with ABL kinase inhibitors, Ph-like ALL need to be treated with multiple inhibitors to induce the same effect. Here we show that a combination of JAK, PI3K and SRC-kinase inhibitors is needed to prevent reactivation of signaling pathways and to induce an apoptotic effect that may be clinically relevant. DisclosuresHurtz:Incyte: Research Funding. Tasian:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Research Funding. Ruggeri:Incyte Corporation: Employment, Equity Ownership. Stubbs:Incyte Corporation: Employment, Equity Ownership. Perl:Daiichi Sankyo: Consultancy; Asana Biosciences: Other: Scientific advisory board; Arog Pharmaceuticals: Consultancy; Novartis: Other: Advisory Board; Seattle Genetics: Other: Advisory board; Astellas: Consultancy; Pfizer: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding.

Transcriptional circuits in B cell transformation.

Loss of IKAROS in committed B cell precursors causes a block in differentiation while at the same time augments aberrant cellular properties, such as bone marrow stromal adhesion, self-renewal and resistance to glucocorticoid-mediated cell death. B cell acute lymphoblastic leukaemias originating from these early stages of B cell differentiation and associated with IKAROS mutations share a high-risk cellular phenotype suggesting that deregulation of IKAROS-based mechanisms cause a highly malignant disease process. Recent studies show that IKAROS is critical for the activity of super-enhancers at genes required for pre-B cell receptor (BCR) signalling and differentiation, working either downstream of or in parallel with B cell master regulators such as EBF1 and PAX5. IKAROS also directly represses a cryptic regulatory network of transcription factors prevalent in mesenchymal and epithelial precursors that includes YAP1, TEAD1/2, LHX2 and LMO2, and their targets, which are not normally expressed in lymphocytes. IKAROS prevents not only expression of these 'extra-lineage' transcription factors but also their cooperation with endogenous B cell master regulators, such as EBF1 and PAX5, leading to the formation of a de novo for lymphocytes super-enhancer network. IKAROS coordinates with the Polycomb repression complex (PRC2) to provide stable repression of associated genes during B cell development. However, induction of regulatory factors normally repressed by IKAROS starts a feed-forward loop that activates de-novo enhancers and elevates them to super-enhancer status, thereby diminishing PRC2 repression and awakening aberrant epithelial-like cell properties in B cell precursors. Insight into IKAROS-based transcriptional circuits not only sets new paradigms for cell differentiation but also provides new approaches for classifying and treating high-risk human B-ALL that originates from these early stages of B cell differentiation.

Exploring Pre-B Cell Receptor Signalling As a Therapeutic Target in Acute Lymphoblastic Leukaemia

Introduction:Acute lymphoblastic leukaemia (ALL) is a clonal disorder of developing lymphocytes and is the most common malignancy in children and adolescents. While cure rates are high, treatment is associated with significant morbidity and relapsed ALL remains one of the leading causes of cancer-related deaths in children. New, less toxic therapies are clearly needed for refractory ALL.There are a number of lines of evidence to suggest that ALL cells hijack components of precursor-B cell receptor (Pre-BCR) signalling and this dependency may be amenable to therapeutic exploitation. There are a number of tyrosine kinase inhibitors (TKIs) targeting Pre-BCR signalling that are showing great promise in the clinic for other leukemia subtypes which warrant preclinical evaluation in childhood ALL.[1] These include CAL-101 (PI3K-δ inhibitor), Ibrutinib (BTK inhibitor), Fostamatinib R406 (SYK inhibitor) and Dasatinib (BCR-ABL/SRC inhibitor).Methods:TKIs were evaluated in ALL cells, including cell lines (PreB 697 and its glucocorticoid resistant descendant, R3F9; Nalm-6 and Reh) and 25 patient derived xenograft samples (PDX) from 12 predominantly high risk/relapse children ALLs. Resazurin was used to assess cell viability. Flow cytometry was used to detect Pre-BCR expression (µHc, Vpreb and λ5) and functionality using a Calcium flux assay. Phospho-flow cytometry was performed to monitor constitutive phosphorylation and response to Pre-BCR activation and to assess pharmacodynamic drug action (p-AKT, p-BLNK, p-BTK, p-SYK, p-ERK, and p-PLC-ϒ2). GILZ expression was measured by RQ-PCR. Cell cycle and apoptosis were determined by flow cytometry using Propidium Iodide and Annexin V staining.Results:ALL cell lines were resistant to CAL-101 (mean GI50 52.08 µM, range 25 µM-77.83 µM) and Ibrutinib (mean GI50 15.9 µM, range 11.47 µM-18.3 µM). However, modest sensitivity was seen to R406 (mean GI50 4.32 µM, range 2.88 µM-5.83 µM) and Dasatinib (mean GI50 5.33 µM, range 2.45 µM-12.5 µM). CAL-101 and Dasatinib were shown to be cytostatic, causing G1 arrest but no significant apoptosis, while Ibrutinib and R406 were associated with cell cycle arrest and significant apoptosis after 72 hours incubation with GI50 concentrations (16.81±1.71 % and 31.34±5.78 % apoptosis, respectively). Pre-B receptor positive cells were more sensitive to Dasatinib and Fostamatinib. Pharmacodynamic assessment using Phospho-flow cytometry and Western blotting showed inhibition of the relevant targets at the GI50 concentrations. PDX ALL cells were generally more sensitive than the cell lines; CAL-101 (mean GI50 25.56 µM, range 76 nM-100 µM; 2 out of 12 patient samples <2µM); Ibrutinib (mean GI50 14.23 µM, range 490 nM-100 µM; 3 out of 12 patient samples <5µM); R406 (mean GI50 11.52 µM and range 56 nM-25 µM, 4 out of 12 patient samples <4µM); Dasatinib (mean GI50 25.56 µM, range 76 nM-100 µM; 3 out of 12 patient samples <0.5µM). ALLs sensitive to Dasatinib were Ph+ or Pre-BCR positive and the latter were also sensitive to Fostamatinib.Synergism was seen after co-treatment of TKIs with the glucocorticoid (GC), Dexamethasone (CAL-101 CI mean 0.7, range 0.056-1.34; Ibrutinib CI mean 0.71, range 0.41-0.97; R406 CI mean 0.27, range 0.1-0.6 and Dasatinib CI mean 0.63, range 0.14-1.29). No synergism was observed in the glucocorticoid receptor negative, Reh cell line. For R406 and Dasatinib, co- exposure was strongly synergistic and was associated with increased apoptosis in PreB 697 and the GC resistant lines, Nalm-6 and R3F9. Synergism was associated with a significant increase in expression of the GR target, GILZ and an enhanced downregulation of R406 and Dasatinib targets (p-SYK for R406 and p-BTK, p-SYK for Dasatinib).Conclusion:We have identified significant sensitivity of TKIs impacting on Pre-BCR signalling in ALL cells at clinically relevant concentrations; Pre-BCR positive ALLs were associated with Dasatinib and Fostamatinib sensitivity; Pre-BCR negative ALL cells were also sensitive to some TKIs, although predictive biomarkers remain to be established. Marked synergism was observed in combination with dexamethasone, even in GC resistant cells. In vivo preclinical confirmation of these data may offer new therapies for refractory ALL.1. Young, R.M. and L.M. Staudt, Targeting pathological B cell receptor signalling in lymphoid malignancies. Nature Reviews Drug Discovery, 2013. 12(3): p. 229-243. DisclosuresNo relevant conflicts of interest to declare.

Science Signaling Podcast for 29 November 2016: Pre-B cell receptor signaling in leukemia.

This Podcast features an interview with Bridget Wilson, author of a Research Article that appears in the 29 November 2016 issue of Science Signaling, about pre-B cell receptor (pre-BCR) signaling in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Signaling through the pre-BCR, an immature form of the BCR, promotes the survival of B cell progenitors and has been implicated in the pathology of BCP-ALL. Erasmus et al found that pre-BCRs formed transient homomeric complexes that correlated with pro-survival signaling. Preventing homotypic interactions between pre-BCRs sensitized B cells to chemotherapeutic agents, suggesting that interfering with such interactions may improve the efficacy of existing chemotherapies for BCP-ALL.Listen to Podcast.

Charged Amino Acid-rich Leucine Zipper-1 (Crlz-1) as a Target of Wnt Signaling Pathway Controls Pre-B Cell Proliferation by Affecting Runx/CBFβ-targeted VpreB and λ5 Genes

The proliferation of pre-B cells is known to further increase the clonal diversity of B cells at the stage of pre-B cells by allowing the same rearranged heavy chains to combine with differently rearranged light chains in a subsequent developmental stage. Crlz-1 (charged amino acid-rich leucine zipper-1) was found to control this proliferation of pre-B cells by working as a Wnt (wingless-related mouse mammary tumor virus integration site) target gene in these cells. Mechanistically, Crlz-1 protein functioned by mobilizing cytoplasmic CBFβ (core binding factor β) into the nucleus to allow Runx (runt-related transcription factor)/CBFβ heterodimerization. Runx/CBFβ then turned on its target genes such as EBF (early B cell factor), VpreB, and λ5 and thereby pre-B cell receptor signaling, leading to the expression of cyclins D2 and D3 Actually, the proliferative function of Crlz-1 was demonstrated by not only Crlz-1 or β-catenin knockdown but also Crlz-1 overexpression. Furthermore, the mechanistic view that the proliferative function of Crlz-1 is caused by relaying Wnt/β-catenin to pre-B cell receptor signaling pathways through the regulation of Runx/CBFβ heterodimerization was also verified by employing niclosamide, XAV939, and LiCl as Wnt inhibitors and activator, respectively.

PTEN opposes negative selection and enables oncogenic transformation of pre-B cells.

PTEN is a negative regulator of PI3K-AKT signaling and a potent tumor suppressor in many types of cancer. To test a tumor suppressive role of PTEN in pre-B acute lymphoblastic leukemia (ALL), we induced Cre-mediated deletion of Pten in mouse models of pre-B ALL. In contrast to its role as a tumor suppressor in other cancers, loss of one or both alleles of Pten caused rapid cell death of pre-B ALL cells and was sufficient to clear transplant recipient mice of leukemia. Small molecule inhibition of PTEN in human pre-B ALL cells resulted in AKT hyperactivation, p53 checkpoint activation and cell death. Loss of PTEN function in pre-B ALL cells was functionally equivalent to acute activation of autoreactive pre-BCR signaling, which engaged a deletional checkpoint for removal of autoreactive B cells. We propose that targeted inhibition of PTEN and hyperactivation of AKT triggers a checkpoint for elimination of autoreactive B cells and represents a new strategy to overcome drug-resistance in human ALL.

CD25 (IL2RA) Orchestrates Negative Feedback Control and Stabilizes Oncogenic Signaling Strength in Acute Lymphoblastic Leukemia

Background and hypothesis: CD25 (IL2RA) represents the α chain of the interleukin 2 receptor on T cells and plays an important role in the maintenance of regulatory T (Treg) cells, hence preventing T cell autoimmunity. In a comprehensive gene expression analysis, we found that CD25 is specifically upregulated by pre-B cell receptor (pre-BCR) signaling during early B cell development and oncogenic tyrosine kinase that mimic pre-BCR signaling (e.g. in Ph+ ALL and Ph-like ALL). In adults with Ph+ ALL (ECOG; MDACC) and children with Ph-like ALL (P9906) patients with CD25 expression at the time of diagnosis have a particularly poor outcome (n=416; P =0.005). For these reasons, we studied the function of CD25 in B cell development and leukemia in a series of genetic experiments.Results: Unlike T cells, CD25 (IL2RA) does not function as IL2 receptor chain in B cells and B-lineage ALL: CD25 expressed on B-lineage cells did not pair with IL2Rb and g-chains and was not responsive to IL2. Il2ra-/- B cells were arrested at the pre-B cell stage with hyperactive pre-BCR downstream signaling including SRC, BTK and ERK. In the presence of CD25, Il2ra+/+ B cells responded to engagement of the pre-BCR with phosphorylation of pre-BCR downstream tyrosine kinases and coordinated release of Ca2+ from cytoplasmic stores. In the absence of CD25 (Il2ra-/-), the pre-BCR signals autonomously, resulting in uncoordinated Ca2+ oscillations of variable duration. While CD25 does not function as IL2 receptor chain in B cells, it coordinates pre-BCR-dependent signal transduction and regulates its intensity.The pre-BCR related tyrosine kinase BTK is phosphorylated by BCR-ABL1 in Ph+ ALL and other tyrosine kinase oncogenes in Ph-like ALL (Chen et al., 2015). Interestingly, overexpression of a constitutively active form of BTK resulted in strong upregulation of CD25 surface expression. Conversely, the BTK-inhibitor ibrutinib abolished CD25 expression suggesting that feedback control between pre-BCR signaling and CD25 requires BTK.The ability of CD25 to stabilize oncogenic signaling strength in Ph+ ALL and Ph-like ALL was important for leukemia-initiation and development of fatal disease. In the absence of CD25, Il2ra-/- ALL cells showed impaired proliferation and colony formation. Serial transplantation experiments revealed a profound defect of Il2ra-/- ALL cells to initiate leukemia. 100-times more cells were required to cause fatal disease. In addition, CD25 expression mediated drug-resistance in ALL cells: In patient-derived pre-B ALL cells with heterogeneous CD25 expression, vincristine selectively induced apoptosis in CD25Low cells but spared CD25High ALL cells. Combination with an anti-CD25 immunotoxin efficiently eradiated CD25High leukemia cells and sensitized the ALL cell population to treatment with vincristine.To elucidate the mechanism of how CD25 coordinates negative feedback control of pre-BCR signaling or its oncogenic mimics, we focused on its short (13aa) cytoplasmic tail, which includes two phosphorylation sites (S268 and T271) that are known substrates for serine/threonine protein kinase, PKCα. To identify cytoplasmic interaction partners of CD25, we overexpressed a Flag-tagged truncated form of CD25 including a myristoylation signal for constitutive membrane localization, transmembrane domain and cytoplasmic tail. Immunoprecipitation (IP; Flag) followed by 2D mass spectrometry revealed strong interactions of PP2A with cytoplasmic tail of CD25. Western blots showed additional strong interactions of the cytoplasmic tail of CD25 with inhibitory phosphatases PTEN, SHP1 and SHIP1. Importantly, reconstitution of myristoylated CD25 tail but not a mutant construct lacking the serine/threonine motif (S268A/T271A) rescued proliferation and survival defects of Il2ra-/- ALL cells.Conclusion: We identified CD25 as a surface receptor that mediates membrane recruitment of PP2A, PTEN, SHP1 and SHIP1, which balances fluctuations in signaling output from a pre-B cell receptor or its oncogenic mimic in ALL cells (e.g. BCR-ABL1 in Ph+ ALL). We propose that CD25-mediated negative feedback control stabilizes oncogenic tyrosine kinase signaling and mediates drug-resistance in Ph+ ALL and Ph-like ALL cells. Targeted inhibition using CD25-directed immunotoxins may be useful in new approaches to overcome drug-resistance in Ph+ ALL and Ph-like ALL. DisclosuresNo relevant conflicts of interest to declare.

Pre-B-cell Receptor Signaling Is a Therapeutic Target in Pre-B ALL.

Abstract Tonic pre–B-cell receptor (pre-BCR) signaling identifies a unique subset of human ALL.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Self-Enforcing Feedback Activation between BCL6 and Pre-B Cell Receptor Signaling Defines a Distinct Subtype of Acute Lymphoblastic Leukemia

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.