ALBERT

Description: Introduction: Children with Down syndrome (DS) have a 10-20 fold increased risk of acute lymphoblastic leukemia (ALL) and suffer significantly poorer outcomes due to increased relapse and treatment-related mortality. DS-ALL exhibits a distinct spectrum of cytogenetic alterations, with a lower incidence of common recurrent ALL alterations, a higher incidence of alterations involving CRLF2 and JAK2, and a higher proportion of cases lacking a known oncogenic driver compared to non-DS ALL. We hypothesized that identifying genetic drivers in DS-ALL will facilitate improved risk-based treatment stratification and lead to novel therapeutic targets that may be harnessed to improve outcomes in this vulnerable population. Methods: We interrogated diagnostic tumor (bone marrow or peripheral blood) and matched remission ("normal") blood samples from a cohort of 63 DS-ALL cases (65% male; median age 5 years, range 1-21 years) obtained from Texas Children's Cancer Center and the Children's Oncology Group. Samples were selected to include ~1/3 CRLF2-rearranged (CRLF2-R) and ~2/3 CRLF2-wild-type (CRLF2-WT) cases. Whole exome sequencing (WES) and copy number analysis using the OmniExpress array were performed on both tumor and normal DNA and transcriptome sequencing (RNAseq) was conducted on tumor RNA, all using the Illumina platform and existing informatic pipelines at the Baylor College of Medicine Human Genome Sequencing Center. A median of 〉97% of targeted bases with at least 20x coverage (range 93.5-98.1%) was observed for WES, and an average of 55.4 million paired reads were generated per RNAseq. Results: Analysis of RNAseq data (n=60) using deFuse and SOAPfuse algorithms revealed known ALL-associated gene rearrangements in 37/60 (62%) of cases, including CRLF2-R (21/60, 35%) involving P2RY8 (n=17) or IGH (n=4); ETV6-RUNX1 fusions (14/60, 23%) and TCF3 fusions (PBX1 x 2 and a novel in-frame fusion with FLI1 x 1). WES (n=62) identified a median of 19 nonsilent somatic mutations per case (range 6-228). We did not find evidence of somatic or germline mismatch repair gene mutations in the 3 cases with 〉50 somatic mutations. WES findings included recurrent activating hotspot mutations in JAK2 (predominantly p.R683G/S) in 14 cases (23%) and CRLF2 (p.F232C) in 2 cases, all of which also had CRLF2-R. Somatic RAS pathway mutations (13 KRAS, 7 NRAS, and 2 PTPN11) were found in 20/62 cases (32%), 4 CRLF2-R and 16 CRLF2-WT, including 2 cases also harboring JAK2 activating mutations. Recurrent mutations in epigenetic or chromatin remodeling genes (e.g. KMT2C, SETD2, KDM6A, WHSC1, DOT1L) were observed in ~25% of patients, as were mutations in other known ALL genes (e.g. FLT3, IL7R, IKZF1, PAX5). Interestingly, inactivating mutations in the deubiquitinase USP9X, also recently reported in acute promyelocytic leukemia, were identified in 5/62 (8%) cases. Evaluation for internal tandem duplications (ITDs) demonstrated typical in-frame events in FLT3 (n=2) and an out-of-frame ITD in SETD2. Copy number analysis revealed frequent losses in cell cycle genes (CDKN2A/B, BTG1, RB1) and other known B-cell development genes (IKZF1, PAX5). Other putative candidate alterations detected by RNAseq, WES, and array are currently being validated by orthogonal platforms. We observed multiple subclonal driver events in 〉10% of patients, including one case with CRLF2-R and mutations in CRLF2, JAK2, KRAS, and NRAS; and a second case with CRLF2 and ETV6-RUNX1 rearrangements and a PTPN11 hotspot mutation. Six cases exhibited 2 different JAK2, KRAS, or NRAS mutations: in all four cases where evaluation was feasible, these mutations were present on different sequencing reads, suggesting the existence of distinct subclones. Clinical features (gender, age, initial white blood count) did not appear to co-cluster with any of the recurrent alterations. Conclusions: These findings detail the landscape of genomic and transcriptomic alterations in the largest cohort of DS-ALL comprehensively characterized to date. Our data confirm the frequency and occasional polyclonal nature of JAK2 and RAS pathway mutations, and demonstrate that mutations in these pathways are not mutually exclusive. We also identified several novel, recurrent mutations and fusions. Future analyses of additional cohorts will be needed to assess recurrence frequency and prognostic impact of these alterations. Disclosures Schore: Baxalta: Honoraria; Millennium Pharmaceuticals, Inc: Research Funding; Onyx/Amgen: Research Funding; Merck: Research Funding.

Description: Introduction: Children with Down syndrome (DS) are 10-20 times more likely than children without DS to develop acute lymphoblastic leukemia (ALL), and they demonstrate a distinctive spectrum of genetic alterations. Approximately 50% of DS-ALL cases demonstrate CRLF2 rearrangements (CRLF2-R), an approximately 10-fold higher frequency than in non-DS ALL. We sought to identify the functional basis for the increased incidence of ALL, and specifically CRLF2-R ALL, in children with DS. Methods: We created retroviral vectors which induce overexpression of CRLF2 and green fluorescent protein (GFP) for transduction into bone marrow (BM) cells isolated from the Dp16(1)Yey (Dp16) mouse model of DS, which is trisomic for the approximately 115 human chromosome 21 gene orthologs present on mouse chromosome 16. Transduced BM cells from Dp16 and wild-type (WT) control mice were co-cultured with OP9 stromal cells for one week to promote B-lymphoid lineage development, and then characterized by flow cytometric Hardy fraction analysis, or grown in B-lymphoid-promoting methylcellulose medium for colony growth assays. Results: We achieved efficient transduction (80-95%) of Dp16 and WT BM enriched for hematopoietic stem cells (HSCs) with CRLF2-GFP+ and control GFP+ viruses. Following OP9 co-culture, transduced HSCs were characterized by Hardy fraction analysis. CRLF2-GFP+ Dp16 lymphoid cells demonstrated significantly higher percentages of immature Fraction A (pre-pro-B) cells compared with GFP+ Dp16 cells (39.9% vs 15.7%, p=0.004, Fig. 1A). This CRLF2-GFP-induced immature immunophenotype was more pronounced in Dp16 versus WT HSCs, with a significantly higher percentage of Fraction A cells (39.9% in Dp16 vs 24.0% in WT, p=0.0002) and a significantly lower percentage of more mature Fraction B (pro-B) cells (24.3% in Dp16 vs 49.1% in WT, p=0.02, Fig. 1A,B). In methylcellulose colony assays, CRLF2-GFP+ Dp16 cells yielded a 36-fold increase in B cell colonies compared to GFP+ Dp16 cells (Fig 1C). Again, the effect of CRLF2 transduction was magnified in the Dp16 versus WT background. CRLF2-GFP+ WT cells demonstrated only a 2.9-fold increase in B cell colonies (Fig 1C). Conclusions: Here we demonstrate that CRLF2 overexpression results in a more immature B-lineage immunophenotype and increased lymphoid colony growth in vitro, and that these effects are significantly greater in a murine DS versus WT genetic background. Experiments to investigate the pathways involved and to evaluate these effects in vivo are ongoing. This work provides functional evidence of the enhanced leukemogenicity of CRLF2 overexpression in DS-ALL, and creates a tractable model system for additional future genetic studies. Disclosures No relevant conflicts of interest to declare.

Description: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and relapsed ALL is a leading cause of cancer-related death in children. While advances in frontline therapy have led to an 85% cure rate, relapsed ALL patients face a dismal prognosis necessitating identification of novel targets and development of alternative therapies. Amongst the two sub types of ALL, T-cell acute lymphoblastic leukemia (T-ALL) occurs less frequently but T-ALL patients have worse prognosis and higher rate of relapse. Our group discovered that the transcription factor KLF4 is heavily repressed via DNA methylation coinciding with aberrant activation of MAP2K7 and the MAP2K7 pathway (Shen et al. Leukemia, 2017). Additionally, we demonstrated, as a proof of principle, that pharmacological inhibition of JNK, the only downstream target of MAP2K7, possesses anti-leukemic properties against T-ALL but presented obstacles in terms of low potency and off-target effects. In order to overcome these limitations, we hypothesize that direct pharmacological inhibition of MAP2K7 improve specificity for targeted approach to T-ALL therapeutics. To examine this hypothesis, we explore the anti-leukemic effects of the MAP2K7 pathway inhibitor, 5Z-7-oxozeaenol, against T-ALL cells because it has been shown to covalently react with a unique cysteine-218 in the ATP binding pocket of MAP2K7. We found that T-ALL cell lines exhibit increased cytotoxic sensitivity to 5Z-7-oxozeaenol (IC50 ranging 0.2-1.1 µM) compared to a non-leukemic control (1.5 µM), and more potent than JNK inhibitor IC50 of 5µM. Additionally, 5Z-7-oxozeaenol reduces the amount of phospho-JNK, in a dose dependent manner, indicative of MAP2K7 pathway inhibition. We purified MAP2K7 protein to directly address specificity of kinase inhibition and found that 5Z-7-oxozeaenol inhibits enzymatic activity in vitro of MAP2K7. In addition, 5Z-7-oxozeaenol also inhibited TAK1, which is the MAP3K upstream of MAP2K7. In a panel of T-ALL cell lines, 5Z-7-oxozeaenol treatment induced apoptosis in MOLT3, Jurkat, and KOPTK1 T-ALL cell lines. Although P12-Ichikawa, RPMI-8402, DND-41, and ALL-SIL T-ALL cell lines underwent cell cycle arrest evidenced by a reduction in percentage of S/G2/M phase cells and increase in percentage of G0 phase cells, further increasing 5Z-7-oxozeaenol dosage proved sufficient for induction of apoptosis based on increase of caspase-3 and caspase-7 cleavage. Through reverse-phase protein array analysis we identified reduced expression of several cell cycle regulator proteins, including CDC25C, Cyclin B1, Cyclin D3, and CDC2 resulting from 5Z-7-oxozeaenol treatment. Conversely, we detected increased expression of cleaved caspase-3 and caspase-7, particularly in MOLT3 and Jurkat cell lines treated with MAP2K7 inhibitor. Immunoblot analysis revealed that 5Z-7-oxozeaenol inhibits the MAP2K7 signaling pathway and induces cell cycle arrest and apoptosis in T-ALL cells. Based on these findings, we demonstrated that 5Z-7-oxozeaenol induces cell cycle arrest and apoptosis in T-ALL cells through inhibition of the MAP2K7 pathway, suggesting that MAP2K7 represents a novel pharmacological target for the development of targeted therapy for high-risk patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: RAS-activating mutations are common in both childhood B- and T-acute lymphoblastic leukemia (ALL). Prior studies of B- and T-ALL have shown that RAS mutations become enriched during treatment in minimum residual disease-positive cases, are associated with a poor glucocorticoid response, and are associated with inferior survival in relapsed disease. There is a need for more tractable preclinical models of RAS mutation-driven B- and T-ALL. Currently, most genetically-engineered mouse models generated to study KRASG12D-driven ALL have a high latency, low penetrance, and/or necessitate the use of multiple technical manipulations, which can yield inconsistent results. Here, we set out to generate a more efficient and penetrant mouse model of KRASG12D-driven B-ALL or T-ALL. Methods:We utilized KRASLSL-G12D/+ mice, which carry KRASG12D preceded by a Lox-Stop-Lox (LSL) site. KRASLSL-G12D/+ males were crossed with Mb1Cre/+ females, which express Cre in most (68-90%) B lineage cells and in a small percentage (