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: Background Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and is characterized by the acquisition of recurrent genetic aberrations, which include chromosomal translocations, submicroscopic deletions and point mutations. The t(12;21) ETV6-RUNX1 translocation is present in about 25% of all pediatric B-cell precursor ALL (BCP-ALL) cases and represents an early genetic event, which can already be detected in utero. However, ETV6-RUNX1 expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Results Using extensive genomic profiling of pediatric BCP-ALL we and others have demonstrated that several recurrent gene deletions can be found in ETV6-RUNX1-positive leukemia. These include focal deletions affecting the B-cell translocation gene 1 (BTG1), which is a member of the BTG/Tob family of anti-proliferation genes. BTG1 deletions are present in 9% of all BCP-ALL cases, but appear to be specifically enriched (20%) in ETV6-RUNX1-positive leukemia. BTG1 protein displays no intrinsic enzymatic activity but may act by recruiting effector molecules like protein arginine methyltransferase 1 (PRMT1) to specific transcription factors, including RUNX1 and ATF4. To investigate whether loss of BTG1 function cooperates with ETV6-RUNX1 in leukemic transformation we developed an in vitro transformation assay. To this end, primitive fetal liver progenitors (FLPs) were purified as c-Kit+ Ter119- cells from fetal livers of embryonic day 13.5 (E13.5) of C57Bl6/J wild-type and Btg1-/- mice. After transduction with control pMSCV-IRES-GFP (pMIG) and pMIG-ETV6-RUNX1 retrovirus, cells were serially replated in methylcellulose or liquid culture in the presence of cytokines SCF, FLT3L and IL-7. We observed a proliferative growth advantage of ETV6-RUNX1 over control virus, and in BTG1-deficient FLPs as compared to wild-type FLPs. Notably, the proliferative advantage of BTG1-deficient FLPs was even further enhanced by co-expression of ETV6-RUNX1. By immunoprecipitation from FLPs, we could demonstrate that endogenous PRMT1 binds to ETV6-RUNX1, and this interaction is lost in BTG1-deficient FLPs, arguing that BTG1 is required for the interaction between PRMT1 and ETV6-RUNX1. In summary, our data indicate that loss of BTG1 function promotes leukemic transformation induced by oncogenic fusion protein ETV6-RUNX1, which implies that BTG1 gene deletions can act as a cooperating event in ETV6-RUNX1-driven leukemogenesis. Disclosures: No relevant conflicts of interest to declare.