ALBERT

Description: Abstract 1104 Poster Board I-126 Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse. Disclosures No relevant conflicts of interest to declare.

Description: The phosphoinositide 3-kinase (PI3K)-AKT/PKB signal transduction cascade is an evolutionarily conserved pathway regulating the FOXO subclass of Forkhead transcription factors (FOXO1, FOXO3a and FOXO4) downstream of insulin, cytokine and growth factor receptors. When PI3K phospholipid kinase activity is low, FOXO proteins localize to the nucleus where they induce expression of genes that promote programmed cell death (FasL, Bim, TRAIL), inhibit cell cycle progression (p27KIP1, Rb related family member p130), facilitate DNA repair (GADD45) and protect against oxidative damage (MnSOD). Following receptor stimulation, increased levels of PtdIns(3,4,5)P3 are generated at the plasma membrane resulting in PDK1-mediated activation of the serine/threonine kinase AKT/PKB. Subsequent phosphorylation of FOXO proteins by AKT/PKB results in their active sequestration in the cytoplasm through binding to 14-3-3 proteins, thereby blocking their action as transcription factors. Recently, it has become evident that FOXO proteins can also be regulated through other signaling pathways. One example is phosphorylation and activation of FOXO4 by stress kinases JNK/SAPK in response to oxidative stress (Essers et al., 2004; EMBO J. 23:4802–12). Here, we report that p38 mitogen activated protein kinases (MAPK) negatively regulate FOXO transcription factors downstream of FLT3 receptor tyrosine kinase signaling. Stimulation of human wild-type FLT3 receptor with FLT3 ligand, or expression of constitutively active mutants FLT3-ITD or FLT3-D835Y in Ba/F3 cells resulted in activation of p38 MAPK and phosphorylation of its known substrate ATF2 on threonine residue 71 (Thr71). Transient luciferase reporter assays showed that expression of a constitutively active MKK3 or MKK6, upstream activators of p38 MAPK, inhibited FOXO-induced gene transcription. The p38 MAPK inhibitors SB202190 and SB203580 as well as dominant-negative p38 MAPK blocked FLT3-mediated inhibition of FOXO transcription activation, suggesting p38 MAPK is both necessary and sufficient for FOXO regulation. In vitro kinase assays demonstrated that FOXO proteins are directly phosphorylated by p38 MAPK, although different p38 MAPK family members (p38α, p38β 2, p38γ and p38δ) displayed distinct FOXO protein substrate specificity. In conclusion, our data demonstrate that FOXO proteins are novel substrates of p38 MAP kinases and activation of p38 MAPK through FLT3 receptor signaling represents an important pathway to attenuate FOXO target gene regulation.

Description: Abstract 3458 Poster Board III-346 Background By genome wide profiling we have found that about 10 % of pediatric pre-B ALL cases contain a (single copy) deletion of the B cell translocation gene 1 (BTG1) gene. BTG1 belongs to a family of potential tumor suppressor genes, which include BTG2, BTG3, TOB and TOB2. Proteins encoded by members of this gene family have been implicated in the induction of growth arrest or apoptosis in a variety of model systems. Moreover, BTG1 associates with and regulates the activity of the arginine methyl transferase PRMT1, a coactivator of nuclear receptor-mediated transcription. Hence we hypothesized that loss of BTG1 function, for instance due to deletion, may affect glucocorticoid induced therapy responses in ALL. Results Using RNA interference, we find that loss of BTG1 expression decreases sensitivity of pre-B ALL cells to the apoptosis-inducing effects of synthetic GCs about 10,000 fold (Figure). This acquired GC resistance is accompanied by a greater than 10 fold reduction in GR protein expression as well as a (near complete) loss of GC-induced gene expression. Conversely, re-expression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression. By chromatin immunoprecipitations using anti PRMT1 antibodies we show that PRMT1 is recruited to the GR gene promoter in a BTG1-dependent manner, consistent with a role for this arginine methyl transferase in the regulation of GR-mediated gene expression. Conclusions Together, our results demonstrate the importance of the BTG1/PRMT1 complex in regulating GR-mediated gene expression and reveal how deregulation of the this complex can give rise to GC resistance. Targeting of these coactivators as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC based therapies in ALL as well as other hematological malignancies. Disclosures No relevant conflicts of interest to declare.