ALBERT

Description: Abstract 182 Chromosomal alterations are a hallmark of acute lymphoblastic leukemia (ALL), but many cases lack a recurring cytogenetic abnormality. To identify novel alterations contributing to leukemogenesis, we previously performed genome-wide profiling of genetic alterations in pediatric ALL using single nucleotide polymorphism (SNP) microarrays. This identified a novel focal deletion involving the pseudoautosomal region (PAR1) of Xp/Yp in 15 B-progenitor ALL cases lacking sentinel chromosomal abnormalities, including six of eight cases of ALL associated with Down syndrome (DS-ALL). The deletion involved hematopoietic cytokine receptor genes, including IL3RA and CSF2RA, but due to poor array coverage, it was not possible to define the limits of deletion using SNP array data alone. To characterize this abnormality, we examined an expanded cohort of 329 B-ALL cases, including 22 B-progenitor DS-ALL cases. Strikingly, 12 (55%) DS-ALL cases harbored the PAR1 deletion. Mapping using high density CGH arrays showed the deletion to be identical in each case, and involved a 320kb region extending from intron 1 of the purinergic receptor gene P2RY8 to the promoter of CRLF2 (encoding cytokine receptor like factor 2, or thymic stromal lymphopoietin receptor). The deletion resulted in a novel fusion of the first, non-coding exon of P2RY8 to the entire coding region of CRLF2 in each case. The P2RY8-CRLF2 fusion resulted in elevated expression of CRLF2 detectable by quantitative RT-PCR, and flow cytometric analysis of leukemic cells. One DS-ALL case with elevated CRLF2 expression lacked the PAR1 deletion, but had an IGH@-CRLF2 translocation detected by fluorescence in situ hybridization (FISH). CRLF2 alteration was associated with gain of chromosome X (which was shown by FISH to result in duplication of the PAR1 deletion), deletion of 9p, and the presence of Janus kinase (JAK1 and JAK2) mutations. Ten (53%) of patients with CRLF2 alteration had JAK mutations, compared with two patients lacking CRLF2 abnormalities (P

Description: Abstract 540 Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified common deletions of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. The IKAROS alterations commonly involve coding exons 3–6, resulting in expression of a dominant negative IKAROS isoform, IK6. Moreover, the presence of IKZF1 alterations is associated with poor outcome in BCR-ABL1 ALL. We have previously shown in a retroviral bone marrow transplant model of BCR-ABL1 ALL that Ikzf1 loss results in increased penetrance of leukemia, but the role of IK6 in the pathogenesis of ALL has not been studied. Here, we have examined the effect of the expression of Ik6 in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from C57BL/6 Arf null mice was transduced with MSCV retrovirus coexpressing p185 BCR-ABL1 and luciferase, plated for 8 days to derive pre-B cells, then transduced with MSCV retrovirus expressing either wildtype Ikaros (Ik1-RFP), Ik6-RFP, or empty vector. Expression of Ik1 was not tolerated and resulted in cell death and apoptosis. IK6 expression led to increased proliferation of p185+Arf null cells with reduced sensitivity to the BCR-ABL1 kinase inhibitor dasatinib compared to cells transduced with empty vector. Intracellular phosphosignaling analysis of Crkl phosphorylation demonstrated that this reduced sensitivity to dasatinib was independent of ABL1 inhibition. Gene expression profiling of p185+Arf null-Ik6 cells revealed a gene expression signature similar to that of human BCR-ABL1+ ALL with enrichment of hematopoietic stem cells genes as well as genes involved in B-cell receptor, Notch, and Jak-Stat signaling pathways. To test the role of Ik6 in leukemogenesis and treatment responsiveness in vivo, p185 BCR-ABL1-luciferase Arf null cells were transduced with MSCV retrovirus expressing GFP alone, Ik1-GFP, or Ik6-GFP then transplanted into lethally irradiated C57BL/6 recipients. Expression of Ik6 in vivo led to accelerated tumorigenesis and decreased survival with tumors uniformly of pre-B immunophenotype. Moreover, mice transplanted with Ik6-expressing marrow were less sensitive to dasatinib therapy (10mg/kg QD initiated 7 days post-BMT) compared to control mice (19d vs. 31.5d, p

Description: Abstract 413 Relapsed acute lymphoblastic leukemia (ALL) is a leading cause of death due to disease in young people, but the biologic determinants of treatment failure remain poorly understood. To identify novel sequence mutations contributing to relapsed in ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. The cohort included B-progenitor ALL with high hyperdiploidy (N=3), TCF3-PBX1 (N=1), ETV6-RUNX1 (N=3), rearrangement of MLL (N=3), BCR-ABL1 (N=3), and low hyperdiploid, pseudodiploid, or miscellaneous karyotypes (N=10). This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including mutations in the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 63 diagnosis-relapse cases and 200 acute leukaemia cases that did not relapse found that 19% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase (HAT) CREB-binding protein (CBP). The mutations were either present at diagnosis, acquired at relapse, or duplicated to homozygosity at the time of relapse. Moreover, several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations confer a selective advantage and promote resistance to therapy. The mutations either resulted in truncated alleles or deleterious substitutions in highly conserved residues of the HAT domain. To examine the functional consequences of the mutations, we introduced wild type or mutant Crebbp alleles into Cbp/Ep300flox/flox murine embryonic fibroblasts, (dKO MEFs), and examined histone acetylation, expression of CREBBP target genes, and cellular proliferation. The HAT domain mutations resulted in impaired acetylation of the key Crebbp substrate, H3K18, and resulted in impaired transcriptional regulation of multiple CREBBP targets and pathways, including cAMP, dsRNA and dexamethasone responsive genes. The latter observation suggests that CREBBP mutations may directly result in resistance to corticosteroid therapy, which is a hallmark of high risk ALL. Together, these data these results extend the landscape of genetic alterations in leukemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL. Disclosures: Pui: EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria.

Description: Abstract 410 Collaborative genomic profiling efforts though the National Cancer Institute's TARGET Initiative and the Children's Oncology Group have identified CRLF2 and JAK mutations in a subset of children with high-risk acute lymphoblastic leukemia (ALL), but few biochemical studies have assessed the functional sequelae of these genetic alterations. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor chain, which heterodimerizes with the IL-7 receptor alpha chain (IL-7Rα). Children with high CRLF2-expressing ALL detected by gene expression profiling have high rates of minimal residual disease at end-induction (Day 29), and approximately 70% of these patients ultimately relapse (Harvey et al., Blood 2010). We hypothesize that characterization of aberrant signaling networks in these leukemias will facilitate identification of potential targets for small molecule inhibitor therapies. Using phosphoflow cytometry, we analyzed the phosphorylation status of key signaling molecules after stimulation with TSLP, IL-7, or pervanadate (an irreversible proximal membrane phosphatase inhibitor used as a positive control) in 2 human ALL cell lines with CRLF2 and JAK2 mutations and in 43 fresh or cryopreserved diagnostic primary patient samples, 27 of which overexpressed CRLF2 through P2RY8-CRLF2 fusion or CRLF2-IgH translocation and 16 of which did not have CRLF2 or JAK mutations (controls). Cells were rested in serum-free media for 60 minutes at 37°C, then stimulated with TSLP, IL-7, or pervanadate for 30 minutes to induce signaling. Cells were also exposed to the JAK inhibitor XL019 (Exelixis) for 60 minutes and/or subsequently stimulated with the aforementioned cytokines or pervanadate to determine the effects of JAK inhibition on signaling. Cells were then processed for phosphoflow cytometry according to our previously published methodologies (Kotecha et al., Cancer Cell 2008). High CRLF2-expressing leukemias (n = 27) with or without concomitant JAK mutations demonstrated strong surface staining of the TSLP receptor, as well as CD10, CD19, and CD127 (IL-7Rα). In vitro stimulation of leukemic blasts with TSLP elicited phosphorylation of STAT5 and S6, but not ERK 1/2, in leukemias with JAK and/or CRLF2 alterations. Control leukemias without CRLF2 and JAK mutations (n=16) did not stain for the TSLP receptor, and TSLP stimulation did not elicit phosphosignaling through the JAK/STAT, PI3K, or MAPK pathways. STAT5 and S6 phosphorylation in the high CRLF2-expressing leukemias was further abrogated by in vitro JAK inhibition with XL019. Surprisingly, despite flow cytometric staining for CD127, stimulation with IL-7 did not elicit phosphosignaling through these epitopes in high CRLF2-expressing or control leukemic blasts, although it did predictably phosphorylate STAT5 in control T and non-blast B cells contained within the primary patient leukemia samples. These results suggest that the JAK/STAT and PI3K pathways, but not the MAPK pathway, are involved in TSLP receptor signaling in high CRLF2-expressing ALL +/− JAK mutations and may represent druggable targets. Phosphoflow cytometry is an efficient method of interrogating intracellular signaling at a single-cell level in primary human samples and, furthermore, can be used for rapid identification of patients at time of leukemia diagnosis with high CRLF2-expressing ALL who exhibit the TSLP phosphosignature. Therapy for this subset of high-risk patients might be modified to include a targeted therapeutic (such as a JAK inhibitor) to improve initial treatment responses and, ultimately, to enhance long-term survival. To this end, we have developed a Children's Oncology Group Phase I clinical trial of JAK inhibition for patients with relapsed or refractory leukemias (including those with CRLF2 and JAK mutations) and will validate the use of phosphoflow cytometry and other biologic assays to assess in vivo target inhibition during therapy. We ultimately envision incorporation of JAK inhibitor therapy into a systemic chemotherapy backbone for patients with high CRLF2-expressing ALL. Disclosures: No relevant conflicts of interest to declare.