ALBERT

Description: Background: Acute Lymphoblastic Leukemia (ALL) in infants is characterized by a high incidence (~80%) of chromosomal rearrangements of the Mixed Lineage Leukemia (MLL) gene, fusing the N-terminal portion of MLL to the C-terminal region of one of its translocation partner genes. MLL-rearranged infant ALL patients are challenged by a very poor prognosis (i.e. 30-40% 5-year EFS), hence the need for better risk stratification and improved therapeutic solutions is evident. We recently screened a relatively large cohort (n=109) infant ALL patients (all enrolled in INTERFANT treatment protocols) for the presence of KRAS and NRAS mutations and found that the incidence of such mutations ranges between 14-24%, depending on the type of MLL translocation. Moreover, these mutations were found to represent independent predictors of exceedingly poor prognosis; patients carrying RAS mutations essentially stand no chance in surviving their malignancy, as all RAS mutation-positive MLL-rearranged infant ALL patients deceased within 3 years from diagnosis. Aims: Here we aimed to identify a therapeutic strategy to improve the prognosis of MLL-rearranged infant ALL patients carrying RAS mutations. Methods: For this, 8 small molecule inhibitors against different RAS-pathway components were selected and initially tested for anti-leukemic activity against the MLL-rearranged ALL cell lines SEM and RS4;11 (RASwt) and KOPN8 (RASmut) using MTS cell viability assays. Next, primary MLL-rearranged infant ALL samples (n=20) all carrying MLL translocation t(4;11) (giving rise to the MLL-AF4 fusion protein) either with (n=6) or without (n=14) RAS mutations were exposed to these inhibitors in MTT cytotoxicity assays. In addition, we assessed the RAS activity in RAS mutated and wild-type MLL-rearranged infant ALL cells, and performed immunoblotting analysis of downstream MEK and ERK both in the absence and presence of the MEK inhibitors. Results: We found that the MEK inhibitors MEK162, Selumetinib and Trametinib effectively reduced the viability of KOPN8 cells (RASmut), whereas SEM and RS4;11 cells (RASwt) largely remained unaffected. In line with this, MLL-AF4+ infant ALL patient samples carrying RAS mutations were significantly more sensitive to these MEK inhibitors when compared with patients carrying wild-type RAS genes: LC50 values for MEK162 were 0.04 vs. 26.9 µM (p

Description: BACKGROUND: MLL-rearranged acute lymphoblastic leukemia (MLLr-ALL) in infants (

Description: Abstract 1426 MLL-rearranged acute lymphoblastic leukemia represents a highly aggressive and clinically unfavorable type of childhood leukemia, displaying unique gene expression signatures. Nevertheless, the overwhelming number of differentially expressed genes made it difficult to elucidate the actual “drivers” of the leukemia. However, recent advances demonstrated that MLL fusion proteins recruit the histone methyltransferase DOT1L, leading to H3K79 methylation. Hence, genomic regions displaying aberrant enrichment of H3K79 methylation are prone to mark genes transcriptionally activated by the MLL fusion protein itself. Based on this concept, two independent studies recently identified gene signatures consisting of genes likely to represent direct MLL fusion targets. Yet, functional validation of such genes so far remains unacknowledged. In the present study we confirmed that CDK6 (cyclin-dependent protein kinase 6) represents a direct target of MLL-AF4 in t(4;11)-positive ALL cells. In contrast to its functional homologue CDK4, ChIP-sequencing analysis showed the presence of both MLL and AF4, as well as H3K79 methylation at the CDK6. Moreover, CDK6 mRNA appeared significantly (p

Description: Background and purpose: Acute lymphoblastic leukemia (ALL) in infants (3), nonsense mutations and nonsynonymous missense mutations involving regions conserved between species. All known annotated single nucleotide polymorphisms according to dbSNP build 138 and an in-house database of 1354 sequenced exomes were excluded. Recurrent mutations were validated by Sanger sequencing on an extended infant ALL patient cohort (n=122). Results: The sequenced exomes had an average coverage depth of 102.5 reads per region or 109 per base. Exome sequencing revealed that, after filtering, t(4;11)+ infant ALL patients on average carried 71 variants (range 58-98, including rare germline variants and somatic mutations). To identify recurrent mutations we selected 23 genes in which two or more patients carried a mutation for further validation by Sanger sequencing. Although all 23 genes were successfully validated, no additional patients carrying these mutations were found. However, recurrent secondary mutations within the same functional region as the initial mutations were found in two genes: PXDN and DSP. The average incidences of the PXDN and DSP mutations amongst MLL-rearranged infant ALL patients were 32% and 20%, respectively. Interestingly, MLL-rearranged infant ALL patients carrying a PXDN mutation had a significantly (p=0.013) better prognosis compared with patients not carrying a PXDNmutation, with mean EFS rates of 6.4 vs. 3.2 years, respectively. Both gene products are involved in intracellular interactions, desmosome formation, extracellular matrix consolidation and phagocytosis and no prior associations with ALL have been made before. Conclusion: We identified 23 genes with two or more mutations in our initial exome sequencing cohort. None of these mutations were found in additional patients in our validation cohort. Despite an average of 71 variations per patient, no recurrent mutations were identified and our study provides evidence supporting previous observations that infant ALL has one of the lowest mutation rates observed in human cancer. However, we identified additional mutations within the same functional region as the initial mutation in DSP and PXDN. Interestingly, PXDN mutations show an association with a favorable prognosis in MLL-R patients. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: Infant acute lymphoblastic leukaemia (ALL) is a rare but aggressive malignancy, mainly presenting with chromosomal rearrangements of the MLL (Mixed Lineage Leukaemia) gene locus on 11q23. The majority of these MLL rearrangements involve the translocation partners AF4, AF9 or ENL within the translocation events t(4;11)(q21;q23), t(9;11)(p22;q23) and t(11;19)(q23;p13.3), respectively. The resulting fusion genes, MLL-AF4, MLL-AF9 and MLL-ENL, code for chimeric transcription regulators acting as strong oncogenic drivers, rewriting the epigenetic landscape of the cell and profoundly altering gene expression. Consequently, these cytogenetic lesions define an ALL subtype both biologically and clinically distinct from other subtypes, strongly associated with drug resistance to first-line chemotherapeutics, high relapse rates and a dismal prognosis. Hence, novel treatment strategies which specifically target the underlying molecular pathobiology of this disease are urgently needed. AIMS: Previously, our group performed extensive patient cohort profiling on both transcript and epigenetic level in order to understand the molecular events underlying the disease, and identified histone deacetylase inhibitors (HDACi) as effective therapeutic drugs both in silico and in vitro. The aim of the current study was to elucidate potential molecular mechanisms by which the candidate HDACi Panobinostat is able to target MLL-rearranged ALL (MLLr-ALL) cells, and to confirm its efficacy in vivo using pre-clinical MLLr-ALL xenograft mouse models able to recapitulate the disease phenotype observed in humans. METHODS: Immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were injected intrafemurally with a MLL-AF4+ B-ALL cell line (SEM) genetically modified to express a luciferase reporter. These mice were subsequently either treated with low-dose (1mg/kg) or high-dose (5mg/kg) Panobinostat using a continuous 5-day-on-2-day-off regimen for a period of up to 12 weeks, or they were assigned to a control group and left untreated. Disease onset and progression was monitored using in vivo bioluminescence imaging, and systemic human ALL cell infiltration was determined by multi-colour flow cytometry and histochemistry. In addition, molecular changes induced by Panobinostat exposure in MLLr-ALL and non-MLLr-ALL cell lines were assessed in vitro using immunoblotting and cell death assays. RESULTS: High-dose Panobinostat resulted in a significantly and substantially delayed MLLr-ALL disease onset and progression in NSG mice when compared to controls; this was accompanied by a reduction of the systemic disease burden, as evidenced by significantly lower whole-body luminescence signals and substantially decreased splenomegaly. Furthermore, immunohistochemical and flow cytometric data showed hypocellularity and increased cell death in the BM of xenografted NSG mice treated with Panobinostat when compared to untreated control xenografts. This finding correlated well with in vitro results, where exposure with 5 nM Panobinostat induced cell death in MLLr-ALL cells, but not in non-MLLr ALL cells, as determined by both ANNEXINV/7AAD flow cytometry assays and immunoblotting. In addition, on a molecular level, in vitro exposure with Panobinostat induced histone H3 hyperacetylation in all leukaemic cell lines, but did not affect other histone modification marks investigated such as, i.e., histone H3K4 methylation or histone H3K79 methylation. A notable exception was observed in MLLr-ALL cell lines, where Panobinostat exposure correlated with a reduction in histone H2B ubiquitination, a histone modification recently reported to be pivotal for MLLr leukaemogenesis. Concomitantly, Panobinostat - or more generally - HDACi-mediated loss of H2B ubiquitination might play a role in the observed sensitivity of MLLr-ALL cell towards this drug class. CONCLUSIONS: Both the in vivo and the molecular in vitro results show the HDACi Panobinostat to have promising therapeutic potential against MLLr-ALL. Currently, we are investigating Panobinostat in combination with other epigenetic drugs in xenograft models with primary MLLr-ALL patient material in order to consolidate these observations, and to confirm HDACi as a novel powerful treatment strategy in MLLr-ALL. Disclosures No relevant conflicts of interest to declare.