ALBERT

Description: Background Glucocorticoids (GCs) such as prednisolone and dexamethasone are critical components of multi-agent chemotherapy regimens used in the treatment of acute lymphoblastic leukemia (ALL). Children with ALL are stratified into risk groups based on diagnostic features (i.e. age and cytogenetics) and therapy response. It has been established that the initial response to prednisolone is a major prognostic factor. Moreover, at relapse, de novo or acquired resistance to GCs is common and represents an important determinant in treatment failure. Recent studies performed by us and others have identified IKZF1 gene deletions and mutations as an independent prognostic factor that predicts prognosis and treatment outcome of children with B cell precursor ALL (BCP-ALL). These monoallelic IKZF1 gene deletions either affect the whole gene or may result in expression of dominant-negative IKZF1 isoforms due to intragenic deletions. However, it has not been established whether loss of IKZF1 function directly impacts the response to glucocorticoids. Results We examined whether haplodeficiency for Ikzf1 gene expression in mouse lymphocytes affects glucocorticoid-induced apoptosis. Splenocytes from Ikzf1+/- knockout mice were activated with lipopolysaccharide (LPS) and treated with increasing concentrations of either prednisolone or dexamethasone for 48 hours. B-lymphocytes haplodeficient for IKZF1 showed a significantly enhanced survival after treatment with GCs compared to wild type cells, as measured in an MTS assay and by AnnexinV staining. In case of prednisolone, the inhibitory concentration (IC50) was about ∼200-fold higher in the Ikzf1+/- splenocytes as compared to the wild-type cells. Gene expression analysis revealed that Ikzf1+/- splenocytes displayed lower overall expression levels as well as diminished transcriptional activation of several glucocorticoid receptor (GR)-induced target genes (i.e. Sgk1, Irs2, Zfp36L2). Furthermore, in luciferase reporter assays we established that IKZF1 overexpression enhances GR-mediated transcriptional activation in response to prednisolone. Finally, lentivirus-mediated IKZF1-shRNA expression in Nalm6 cell line, which reduces endogenous IKZF1 protein levels to around 50%, inhibits prednisolone and dexamethasone-induced apoptosis, demonstrating that also in human leukemia cells reduced IKZF1 expression levels protect against GC-induced cell death. In conclusion, our data provide evidence that loss of IKZF1 function mediates resistance to glucocorticoid-induced apoptosis, which may contribute to the poor outcome of IKZF1-deleted BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

Description: In B cell neoplasia, many transcription factors known to be involved in B cell differentiation and commitment, like E2A, EBF1 and PAX5, are frequently targeted by focal deletions, mutations or chromosomal aberrations. Recent studies have shown that the human genes BTG1 and BTG2 are commonly affected by gene alterations in different B cell malignancies, but their role in normal B cell development has not been established. BTG1 and BTG2 can act as transcriptional cofactors through recruitment of the protein arginine N-methyltransferase PRMT1, which mediates arginine methylation of transcription factors, like RUNX1, and on histone 4 arginine 3 (H4R3). Here we report that Btg1 and Btg2 display unique and overlapping functions during mouse B cell development. We observed a reduction in the fraction of B220+ progenitor cells in the bone marrow compartment of the different knockout animals, ranging from a 10% decrease in the Btg2-/-, 20% in Btg1-/- , and 40% in the Btg1-/-;Btg2-/- mice relative to wild-type controls. Deficiency for Btg1, but not Btg2, resulted in reduced outgrowth of IL-7 dependent lymphoid progenitors in methylcellulose, which correlated with a higher fraction of apoptotic cells. Btg2-/- mice showed impaired differentiation at the pre-pro-B, pro-B and pre-B cell stage, while Btg1-deficiency mainly affected later stages of B cell differentiation with reduced numbers of immature B cells. Btg1-/-;Btg2-/- mice displayed additive effects with more significant reduction of B220+ cells predominantly at the pre-B and immature B cell stage. Expression analysis revealed no reduction in the mRNA levels of master regulators E2a, Foxo1, Ebf1 and Pax5 in the absence of Btg1 and Btg2. However, higher expression levels of T cell-specific genes were observed in Btg1-/-;Btg2-/- progenitor B cells, e.g. Cd4, Ikzf2 and Tcf7 (Figure 1), some of which are known to be transcriptional repressed by Ebf1, such as Id2, Gata3, Dtx3l and Notch1. Flow cytometric analyses confirmed increased expression of CD3, CD4 and CD8 markers on CD19+ bone marrow cells lacking Btg1 and Btg2 function. Additionally, we detected enhanced levels of DC, NK and myeloid markers on Btg1-/-;Btg2-/- CD19+ BM cells, indicating that Btg1 and Btg2 repress alternative cell fates during B cell lineage specification, and are required for the maintenance of B cell identity. Biochemical studies showed evidence for a physical association between Ebf1, Btg1/Btg2 and PRMT1. We propose a model in which Btg1 and Btg2 affect the function of the critical B cell transcription factor Ebf1 by recruitment of PRMT1. Figure 1. Aberrant T-lineage expression in progenitor B cells deficient for Btg1 and Btg2. Relative expression levels of Cd4, Runx1, Ikzf2, Tcf7, Id2, Gata3, Notch1 and Dtx3l were determined on cDNA generated from B220+ BM cells of wild-type (WT), Btg1-/-, Btg2-/- and Btg1-/-;Btg2-/- mice by quantitative real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments containing cDNA derived from 2 different biological samples. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Figure 1. Aberrant T-lineage expression in progenitor B cells deficient for Btg1 and Btg2. Relative expression levels of Cd4, Runx1, Ikzf2, Tcf7, Id2, Gata3, Notch1 and Dtx3l were determined on cDNA generated from B220+ BM cells of wild-type (WT), Btg1-/-, Btg2-/- and Btg1-/-;Btg2-/- mice by quantitative real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments containing cDNA derived from 2 different biological samples. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1303 B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common form of cancer in children, characterized by genetic aberrations affecting master regulators of lymphoid differentiation, such as RUNX1, IKZF1, TCF3, and PAX5, as well as tumor suppressor genes that control the cell cycle, including RB1 and CDKN2A. Another gene frequently altered in BCP-ALL is BTG1, which displays highly clustered mono-allelic deletions in childhood BCP-ALL (9%) and adult ALL (6%). The frequency of BTG1 deletions is two- to three-fold higher in ETV6-RUNX1- and BCR-ABL1-positive leukemias. BTG1, and its close homologue BTG2 regulate gene expression, for instance by associating with protein arginine methyltransferase 1 (PRMT1), affecting the activity of a variety of transcription factors, including several nuclear hormone receptors and HoxB9. In addition, BTG1 and BTG2 have been implicated in regulating mRNA stability by interacting with the Ccr4-Not complex. Recent studies have also identified missense point mutations in BTG1 and BTG2 in about 20% of non-Hodgkin lymphomas, arguing that altered function of these genes contributes to B cell malignancies. To investigate a role of BTG1 and BTG2 in B cell development, we studied the phenotype of Btg1 and Btg2 single knockout (KO) and Btg1;Btg2 double KO mice. Animals deficient for either BTG1 or BTG2 displayed a mild B cell phenotype with a moderate reduction of ∼20% in the total amount of B220+ progenitor B cells in bone marrow, while splenic B cells were present at normal frequencies. More detailed analyses revealed that Btg1−/− and Btg2−/− mice both showed a partial block at the pre-pro-B cell stage (Hardy fraction A). Methylcellulose colony assays in the presence of interleukin-7 (IL-7) demonstrated 30% fewer colonies using bone marrow from Btg2−/− mice, whereas 70% fewer colonies were obtained using bone marrow derived from Btg1−/− mice. To assess whether BTG1 and BTG2 fulfill redundant functions during B cell development, we analyzed the phenotype of Btg1−/−;Btg2−/− mice. Hence we observed that the combined loss of BTG1 and BTG2 led to a much stronger block in B cell differentiation, with the majority of progenitor B cells arrested at the pre-pro-B cell stage. In the spleens of these double knockout mice we observed a roughly 50% reduction in B220+ IgM+ B cells, suggesting that these genes act to modify the activity of B lineage transcription factors rather than to fully block their activities. This is consistent with a role for these genes as modifiers of transcriptional activity. Current studies are aimed at defining the molecular targets regulated by BTG1 and BTG2 during early B cell development using RNA sequencing and protein interaction experiments. In conclusion, our data demonstrate that BTG1 and BTG2 act as important regulators of normal B cell differentiation, and that this function might be critical for their role as tumor suppressors in (early) B cell malignancies. Disclosures: No relevant conflicts of interest to declare.