ALBERT

Description: The transcription factor Foxp1 is critical for early B cell development. Despite frequent deregulation of Foxp1 in B cell lymphoma, the physiological functions of Foxp1 in mature B cells remain unknown. Here, we used conditional gene targeting in the B cell lineage and report that Foxp1 disruption in developing and mature B cells results in reduced numbers and frequencies of follicular and B-1 B cells and in impaired antibody production upon T cell-independent immunization in vivo. Moreover, Foxp1-deficient B cells are impaired in survival even though they exhibit an increased capacity to proliferate. Transcriptional analysis identified defective expression of the prosurvival Bcl-2 family gene Bcl2l1 encoding Bcl-xl in Foxp1-deficient B cells, and we identified Foxp1 binding in the regulatory region of Bcl2l1. Transgenic overexpression of Bcl2 rescued the survival defect in Foxp1-deficient mature B cells in vivo and restored peripheral B cell numbers. Thus, our results identify Foxp1 as a physiological regulator of mature B cell survival mediated in part via the control of Bcl-xl expression and imply that this pathway might contribute to the pathogenic function of aberrant Foxp1 expression in lymphoma.

Description: Background B cells are selected at multiple developmental checkpoints for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: either insufficient or hyperactive signaling (e.g. from an autoreactive BCR) results in cell death. Acute lymphoblastic leukemia (ALL) is the most frequent type of cancer in children and typically arises from pre-B cells, a large fraction of which are autoreactive. In ∼25% of patients, ALL is driven by an oncogenic tyrosine kinase (e.g. BCR-ABL1 in Ph+ ALL) and defines the ALL subgroup with the worst clinical outcome. Ph+ ALL cells invariably develop resistance against tyrosine kinase inhibitors (TKI). Here we tested the hypothesis that inherent mechanisms of negative selection to eliminate autoreactive clones with hyperactive pre-BCR signaling are still active in transformed pre-B cells and identified a potential therapeutic target for ALL patients. Results The BCR-ABL1 oncogene mimics a constitutively active pre-BCR and an incremental increase of pre-BCR downstream signaling (ITAM overexpression) was indeed sufficient to induce cell death in Ph+ ALL, but not in normal pre-B cells with low baseline signaling strength. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. Patient-derived Ph+ ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1, CD300A and LAIR1. However, ITAM containing activation receptors like CD79B was absent on the cell surface, and there was point or frame-shift mutation for both CD79A and CD79B. 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 LAIR1 at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p

Description: Abstract 562FN2 Background: The BACH2 (BTB and CNC homology, basic leucine zipper transcription factor 2) transcription factor is required for class-switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of mature germinal center B cells. Interestingly, we and others found that BACH2 is strongly upregulated in BCR-ABL1-transformed acute lymphoblastic leukemia (Ph+ ALL) cells upon treatment with tyrosine kinase inhibitors (TKI). Results: Bach2 mRNA levels are significantly lower in Ph+ ALL (n=72) compared to normal human bone marrow pre-B cells (n=10). We next studied 49 samples pairs from patients with childhood ALL at diagnosis and relapse. In 44 of these sample pairs, the relapse sample showed drastically reduced mRNA levels of Bach2 (p=0.019), suggesting that loss of BACH2 expression is associated with relapse of childhood ALL. Consistent with these findings, an independent study (Children's Oncology Group; NCT00005603) demonstrated that BACH2 mRNA levels in childhood ALL samples at diagnosis negatively correlated with early minimal residual disease (MRD) findings on day 29 (n=207; p

Description: The microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.

Description: Introduction Philadelphia Chromosome positive acute lymphoblastic leukemia (ALL) is associated with particularly poor clinical outcome. Fusion of the ABL1 oncogene to a breakpoint cluster region (BCR) results in constitutively activated BCR-ABL1 tyrosine kinase activity that can be specifically targeted by small molecule tyrosine kinase inhibitors (TKI) such as Imatinib. After initially good clinical response to TKI, BCR-ABL1+ ALL patients invariably relapse into a more aggressive, TKI resistant disease. FOXM1 belongs to the forkhead box transcription factor family and is a key regulator of cell growth by promoting cell cycle progression. While a functional role of the transcription factor FOXM1 in terms of disease progression and drug resistance is well established for solid tumors, it remains elusive whether it has a similar function in ALL. Results We have identified high FOXM1 protein expression levels in patient-derived BCR-ABL1+ ALL samples compared to healthy B cells and B cell precursors. Consistent with this finding, the FOXM1 promoter region was de-methylated in 83 BCR-ABL1+ ALL patient samples compared to normal pre-B cells and Lymphoma samples. We used a mouse-model for BCR-ABL1+ ALL to characterize the role of BCR-ABL1 in the induction of FOXM1 expression: we cultured murine bone marrow B cell precursors in the presence of IL7 and induced transformation with a retroviral BCR-ABL1 expression vector. Consistent with findings for other oncogenes, i.e. activating mutation of Ras, BCR-ABL1 expression increased levels of FOXM1 compared to the normal IL7-dependent pre-B cells. In order to evaluate a potential clinical relevance of FOXM1 expression levels in ALL disease progression, we correlated FOXM1 mRNA levels with clinical outcome. High FOXM1 expression levels correlate with poor clinical outcome of ALL. In addition, the group of Sanders et al. (Genome Biol., 2013) has recently identified a set of 38 genes that are directly regulated by FOXM1 and associated with poor prognosis in breast cancer. 34 of the described 38 FOXM1 target genes were associated with high risk in ALL based on a data set collected by the German ALL-REZ BFM 2002 of the Berlin-Frankfurt-Münster study group (n=60). To study the functional role of FOXM1 in BCR-ABL1+ ALL, we performed experimental genetic deletion experiments using bone-marrow cells derived from Foxm1fl/fl mice. Deletion of Foxm1 decreases cell viability, colony formation, and proliferative capacity in vitro as well as leukemia formation in vivo of BCR-ABL1-driven leukemia. FOXM1-deleted ALL cells revealed a strikingly higher sensitivity towards TKI-treatment compared to the control cells in Imatinib dose-response curves (IC50EV: 420 nM vs IC50 CreERT2: 160 nM) as well as annexin V staining. As FOXM1 is a critical regulator of oxidative responses, we analyzed the intracellular reactive oxygen species (ROS) formation in the presence and absence of FOXM1 in BCR-ABL1 transduced ALL cells and observed consistently higher ROS levels after FOXM1 deletion. As a functional mediator of this effect, we analyzed the expression of the described FOXM1 target and ROS scavenger Catalase and found reduced levels after FOXM1 deletion. We determined Catalase expression in ALL cells after Imatinib treatment and found significant transcriptional upregulation. To evaluate whether this effect is FOXM1-dependent, we treated ALL cells with Imatinib and evaluated Catalase expression in the presence and absence of FOXM1. Only the FOXM1 expressing ALL cells were capable of upregulating Catalase after Imatinib treatment. Single locus chromatin immunoprecipitation (ChIP) analysis of a described binding site of FoxM1 in intron 1 of the catalase gene showed specific binding of FOXM1, albeit low enrichment compared to the positive control cyclin B1. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide and the natural occurring antibiotic Thiostrepton. Both bind FOXM1 and inhibit its function and induced apoptosis in Ph+ ALL. In line with our observation of the genetic deletion, we observed induction of ROS with the treatment of either substance along with a decrease in Catalase expression. Conclusion Taken together, our data identify FOXM1 as a valid therapeutic target for the treatment of TKI sensitive and resistant BCR-ABL1+ ALL – either together with TKI or as a single agent. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2563 Background: SOCS (suppressors of cytokine signaling) are a family of intracellular proteins, including SOCS1–7 and CISH (cytokine induced SH2 domain protein). They play key roles in negative regulation of cytokine signal transduction, including JAK/STAT pathway. While BCR-ABL1 signaling involves STAT5 in Ph+ acute lymphoblastic leukemia (ALL) cells, STAT5 signaling also results in its own feedback inhibition via CISH, SOCS2 and SOCS3. Interestingly, CISH, SOCS2 and SOCS3 are highly expressed in Ph+ALL and represent the focus of this study. Results: Comparing microarray data for human Ph+ ALL (n=15) and normal human pre-B cells (n=8), we found that mRNA levels of SOCS2 and CISH were 10-fold (p

Description: CD4+CD25+ regulatory T cells (Treg) recognize autoantigens and inhibit autoreactive immune responses in a cell contact-dependent manner. In cancer-bearing patients, expansion and functional aberrations of Treg may inhibit immune responses against the tumor. The available evidence suggests that such Treg recognize self antigens expressed by the tumor and argues that induction of anti-tumor T cell responses might be more successful if true tumor-specific rather than lineage-restricted or shared antigens are used for active immunotherapy. Indeed, we have observed a preferential recognition of tumor-individual over shared epitopes by vaccination-induced T cells after immunization of B-NHL patients with recombinant lymphoma idiotype (Bertinetti et al., Cancer Res. 2006). To study this phenomenon in an exemplary fashion, we immunized BALB/c mice with dendritic cells loaded with H-2K-restricted peptides of the immunoglobulin of the A20 lymphoma. A J region-derived peptide served as a model for a shared antigen; a heteroclitic peptide from the CDR3 region represented a tumor-specific antigen. Both peptides bind H-2Kd with similar affinity. Compared to a highly immunogenic influenza HA peptide, the CDR3 peptide was similarly efficient in inducing specific cytotoxic T cells as analyzed by tetramer staining, IFNγ release to peptide stimulation, and in vitro and in vivo cytotoxicity assays with CFSE-labelled, peptide-loaded splenocytes. In contrast, no effector cells were detected with any assay after J immunization. After in vitro restimulation with peptide, however, antigen-specific IFNγ-secreting effector populations were demonstrated for each vaccination, suggesting in vivo inhibition, possibly mediated by Treg, rather than total absence of J-specific T cells. No difference in numbers and the TCR repertoire of CD4+CD25+FoxP3+ cells in the draining lymph node could be detected. However, activation of Treg by J immunization was indicated by potent suppression of antigen-specific splenic effectors compared to CDR3-immunized animals, and by a 4fold higher spontaneous proliferation of FoxP3+ cells from the draining lymph node in vitro. In contrast to CDR3-derived Treg, the addition of J-induced Treg to effector cells resulted in a dose-dependent production of IL-10 in mixed cultures, independently of the antigen specificity of the effectors. Finally, coimmunization with HA and J peptides led to inhibition of the proliferation of HA-specific CD8+ effectors in vivo as demonstrated by adoptive transfer and subsequent flow cytometry analysis of CFSE-labelled TCR-transgenic T cells. This inhibition was absent after coimmunization with HA and CDR3 peptides and could be largely abolished by prior in vivo depletion of Treg with an αCD25 antibody. These data demonstrate in a non-transgenic model that coimmunization with shared and individual, strictly MHC I-restricted tumor antigens leads to a potent inhibition of tumor-specific CD8+ T cells through rapid in situ activation of CD4+FoxP3+ Treg elicited by the shared tumor antigen. It is postulated that these Treg recognize MHC II-restricted self antigens presumably derived from non-neoplastic cells as a consequence of an aborted immune response to the shared antigen. These experiments provide direct evidence that active immunotherapy of malignant tumors exclusively with true tumor-specific antigens has a greater chance of success since the presence of shared antigens will prevent tumor-specific immune responses through Treg activation.

Description: Abstract 291 Background: ITIM (immunoreceptor tyrosine-based inhibition motifs) motifs in the cytoplasmic tail of inhibitory receptors recruit inhibitory phosphatases. Upon ligation, these phosphatases inhibit signal transduction from tyrosine kinases including BCR-ABL1. ITIM-receptors are critical in the control of immune responses of B and T cells. Surprisingly, we found that a number of inhibitory ITIM-receptors are expressed at very high levels on the surface of patient-derived Ph+ acute lymphoblastic leukemia (ALL) cells compared to normal pre-B cells. In this study, three central ITIM-receptors were identified and analyzed for their function in Ph+ALL: PECAM1 (platelet/endothelial cell adhesion molecule 1/CD31), CD300A and LAIR1 (leukocyte-associated immunoglobulin-like receptor 1). Results: Microarray data showed mRNA levels of PECAM1, CD300A and LAIR1 were about 5-fold higher (p

Description: Background & Significance: Pre-B acute lymphoblastic leukemia (ALL) emerges in virtually all cases from B cell precursors that are arrested at the pre-B cell receptor checkpoint. In a gene expression survey of early B cell development, we found specific upregulation of FOXM1 at this developmental stage. FOXM1 belongs to the forkhead box transcription factor family and is a key regulator of cell growth by promoting cell cycle progression and has been implicated in progression of solid tumors. Therefore, we characterized the function and regulation of FOXM1 in normal B cell development as well as in pre-B ALL. Results: First, we verified the upregulation of FOXM1 during B cell development by qRT-PCR on sorted human and murine B cell progenitor populations. Then, we crossed Mb1-Cre tg mice to a Foxm1 conditional knockout mouse model (Foxm1fl/fl) and analyzed the early B cell populations according to the Hardy fractions. Despite the observed high expression of Foxm1 mRNA in fraction C’ and D, Foxm1 deletion did not alter B cell development. In order to investigate a potential role of FOXM1 in transformed B cells, we compared FOXM1 protein levels in patient-derived pre-B ALL samples with healthy B cells and B cell precursors and found 10-60-fold higher expression in the transformed B cell progenitors. To evaluate a potential predictive value of FOXM1 levels in patient-derived ALL samples, we measured FOXM1 mRNA levels at the time of diagnosis which strikingly correlate with risk stratification of ALL (intermediate-risk ALL n=31 vs. high risk ALL n=21; P=7.3e-5; BFM-REZ 2002). To further study the function and regulation of FOXM1, we cultured murine B cell precursors in the presence of IL7 and induced transformation with a retroviral BCR-ABL1 expression vector. BCR-ABL1 expression increased levels of FOXM1 compared to the normal IL7-dependent pre-B cells. Short-term inhibition of BCR-ABL1 did not affected protein levels of FOXM1. However, after 4 days of tyrosine kinase inhibition (TKI) treatment, FOXM1 protein levels were significantly downregulated in a dose-dependent manner. BCL2 overexpression prevented apoptosis induction by TKI but FOXM1 downregulation was retained. In addition, we found evidence that inactivation of FOXO factors by the PI3K/AKT pathway contributes to high FOXM1 expression in Ph+ALL. Overexpression of a constitutively active form of Akt to prevent activation of FOXO factors in the presence of TKI abrogated FOXM1 downregulation. Similarly, BCR-ABL1+ ALL derived from FOXO3a knockout mice prevented TKI-mediated FOXM1 reduction. Overexpression of a constitutively active form of FOXO3a but not FOXO1 significantly reduces levels of FOXM1 expression. In line with this, we found a significant inverse correlation of FOXM1 with FOXO3A mRNA levels in Ph+ ALL patients from the ECOG E2993 trial. However, the requirement of long-term treatment indicates, that, in addition to FOXO3a activation, epigenetic regulation of the FOXM1 promoter downstream of BCR-ABL1 is required. Consistent with this finding, the FOXM1 promoter region was found to be de-methylated in a large fraction of ALL. In order to further study FOXM1 function, we transduced pre-B cells derived from Foxm1fl/fl mice with BCR-ABL1 and with an inducible ERT2-Cre vector. Deletion of Foxm1 in BCR-ABL1-driven leukemia decreases cell viability, colony formation, and proliferative capacity in vitro as well as leukemia formation in vivo. FOXM1-deleted ALL cells revealed a strikingly higher sensitivity towards TKI-treatment compared to the control cells in Imatinib dose-response curves (IC50 EV:420 nM vs IC50 Cre-ERT2: 160 nM) as well as annexin V staining. We identified the ROS scavenger Catalase as a critical target of FOXM1 in mediating this drug resistance. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide and the natural occurring antibiotic Thiostrepton. Both bind FOXM1 and inhibit its function as shown by reduced mRNA expression of FOXM1 target genes (Cyclin B1, PLK1, AURKB) and induced apoptosis in ALL and prolonged survival of patient-derived ALL transplant recipient mice. Conclusion: We have identified a critical function of the transcription factor FOXM1 in mediating proliferation and drug-resistance in B cell lineage ALL, but not in normal B cell progenitors and validated FOXM1 as a therapeutic target in a large fraction of drug-resistant B cell lineage ALL. Disclosures No relevant conflicts of interest to declare.