ALBERT

Description: Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) receptor plays an important role in the pathogenesis of acute myeloid leukemia (AML). A number of small molecule kinase inhibitors are currently proceeding in different phases of clinical trials. As with imatinib in CML, leukemic cells could develop resistance to these RTK inhibitors when used as monotherapy. Mutations in the ATP-binding pocket have been identified through PCR-based mutagenesis screening in Ba/F3-FLT3-ITD cells and selected for growth in the presence of PKC412, or in a resistant Ba/F3-FLT3-ITD cell line developed by cocultured with SU5416. Resistance to PKC412 resulting from the N676K point mutation in the FLT3 kinase domain has been described in a clinical trial patient. Selection of activating Ras mutations has been found in 2 out of 6 FLT3 inhibitor resistant cell lines, but no point mutation of FLT3 kinase domain was found in all 6 resistant cell lines. To further investigate other potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R (IC50: 52 vs 6 nM for the parental MV4-11 cell line), which is also cross resistant to other structurally unrelated inhibitors including SU5416, AG1296 and a FLT3 inhibitor III (MERCK). No point mutation in the kinase domain of FLT3 was found in MV4-11-R cells. Western blot and FACS analysis excluded overexpression of p-FLT3, FLT3 or 3 multidrug resistance related proteins (MDR, MRP1 and LRP) in this resistant line. Gene expression profiling revealed up-regulation of FLT3 ligand (FLT3LG) (2.4 fold) and Survivin (2 fold), while down-regulation of SOCS1, SOCS2, and SOCS3 was observed in MV4-11-R compared to MV4-11 parental cells. Overexpression of FLT3LG and Survivin was also demonstrated at the protein level. Survivin is a unique member of the inhibitor of apoptosis proteins (IAP) family and a known target of the STAT3 pathway. Down-regulation of suppressor of cytokine signaling (SOCS) proteins (negative regulators of STAT pathways) was observed even in the presence of overactivation of the STAT1, STAT3 and STAT5 pathways in the MV4-11-R line. We screened a panel of small molecule inhibitors including a STAT inhibitor (indirubin derivative IDR E804), 3 JAK inhbitors (Tyrene CR4, AG490, and JAK3 Inhibitor II), and a CDK/survivin inhibitor (NU6140). We found that MV4-11-R is most sensitive to IDR E804 (an inhibitor of CDKs and the SRC-STAT3 pathway). The IC50 value of ABT-869 in MV4-11-R was decreased from 52 to 6.2 nM in the presence of 2 nM of IDR E804. Further validation of the therapeutic effect of IDR E804 in combination with ABT-869 in the MV4-11-R mouse xenograft model is ongoing. Targeting Survivin by shRNA and a dominant-negative vector (survivin-T34A) induced MV4-11-R to undergo apoptosis. Taken together, these results demonstrate that overactivation of STAT pathways and overexpression of survivin are the main mechanism of resistance to ABT-869; suggesting that the STAT pathways and survivin could be potential targets for the treatment of patients who develop resistance to FLT3 inhibitors.

Description: Abstract 3519 Pediatric Acute Myeloid Leukemia (AML) offers a unique window into the genesis and progression of hematologic cancers because of its rapid progression and limited confounding factors. As part of the NCI TARGET AML initiative (target.cancer.gov), we report the initial integrative analysis of 58 pediatric AML whole-genome sequences [WGS, performed by Complete Genomics Inc. (CGI)] along with 225 Affymetrix microarrays, clinical cytogenetic and biomarker tests. WGS was performed on matched samples collected at diagnosis and remission from each patient. Candidate variants were identified by CGI and defined as being present only in tumor samples. To focus on the highest confidence variant calls, we developed stringent filters based on a set of 281 true-positive and 44 false-positive verified variants. We identified a total of 629 high-confidence variants in 58 cases (∼ 11 per patient). 99 of these variants occurred in 39 genes (2 to 8 per gene, up to 4 per sample). Analysis of the potential functional consequence of these variants identified 28 as deleterious impacting 17 genes in 21 of the 58 relapsed AMLs, including multiple mutations in AML-associated genes such as KIT, NRAS, PTPN11, and WT1, and several key hematopoietic genes (e.g. IKZF1, GATA2). Using microarray expression data, we identified 1,992 (1,051) genes that were differentially expressed in pediatric AML cases compared to 4 normal bone marrow samples [FDR-adjusted p-value = 0.05 (0.01)]. 249 differentially expressed genes were more than 6 standard deviations away from the control average in more than half of the samples (e.g. WT1, MYCN, miR155), suggesting the existence of a shared set of dysregulated processes across most pediatric AMLs. Network-oriented enrichment analysis using the Bioconductor (http://bioconductor.org) package DEGraph revealed differentially regulated interactions among 3,496 genes, including 1,437 cancer genes and highly enriched interactions involving growth factor/RTK signaling, down-regulated immune processes, and up-regulated transcription and translation. To pinpoint processes specific to AML subtypes, we used the Bioconductor package WGCNA to cluster the 225 microarray expression datasets and align them with clinically-identified cytogenetic and mutation data. We identified five distinct expression clusters. Three of these clusters corresponded to known cytogenetic abnormalities: MLL fusions, t(8;21), and Inv16. The other two clusters were cytogenetically normal, but all members of one cluster carry CEBPA mutations. The expression patterns of cases with t(8;21) and Inv16, while distinct, shared a number of features that distinguished them from samples with MLL abnormalities. For example, in both t(8;21) and Inv16 cases (but not in MLL cases) the fibroblast growth factor (FGF) receptor FGFR1 and the FGF ligand FGF11 were over-expressed compared to control samples. These differences may explain the higher rates of remission associated with Core Binding Factor (CBF) abnormalities. To identify potential relationships between sequence variants and differentially expressed genes, we used the Graphite Bioconductor package to search four publicly available databases (Reactome, NCI PID, KEGG, Biocarta) for known interactions of our candidate deleterious genes. Surprisingly, all but two of our candidate deleterious genes shared many interactors, suggesting they impacted shared processes. The core connected components of the mutation interaction network were all members of the set of dysregulated interactions that we identified by gene expression analysis and included multiple members of well-known pathways implicated in AML and other cancers (e.g. RTK/growth factor signaling, JAK/STAT signaling). Candidate mutations impacted both shared and distinct pathways. Furthermore, within shared pathways, the candidate mutations impacted shared and distinct targets. These findings have important implications for pathway-specific drug targeting. Comparing the interactions of candidate WGS mutations with those of clinically-identified chromosomal abnormalities, a further pattern emerges: CBF and MLL associated gene fusions appeared to impact a different set of genes and processes compared to WGS (presumably second-hit) variants, suggesting complementary roles. This finding has important implications for ongoing research and testing of targeted treatments. Disclosures: No relevant conflicts of interest to declare.

Description: Mutations in the FLT3 gene are among the most common somatic events in AML, with a higher prevalence in adults than in children. The most common activating mutations of FLT3 include internal tandem duplications (FLT3/ITD) in the juxtamembrane domain (JMD) or missense mutations in the tyrosine kinase domain (TKD) at the D835/I836 positions (FLT3/ALM). To date, much of the data on FLT3 mutations has been derived from adult studies and comprehensive sequencing of the FLT3 gene from recent TCGA analysis demonstrated that FLT3 activating mutations were limited to the FLT3/ITD in the JMD and D835/I836 hotspots. As part of the Children's Oncology Group (COG)/NCI TARGET AML initiative, we interrogated the genomic landscape of pediatric AML and identified and verified novel FLT3 activating events that appear to be unique to childhood AML and could provide a target for therapeutic intervention. Whole genome sequencing was performed in a discovery cohort (N=200) followed by validation with targeted exome capture for a total of 799 diagnostic specimens from children treated on contemporary COG trials. In addition to the known FLT3 mutations (FLT3/ITD, N=128 and D835/I836, N=37), we identified novel point mutations (PM; N=49) and novel insertion-deletions (in-dels; N=12). We observed a prevalence of 7.6% of novel PMs and in-dels, in addition to the FLT3/ITD and D835/I836 mutations. The total prevalence of all FLT3 mutations was 28%. In contrast to adult AML, where virtually all non-ITD activating mutations are limited to the D835/I836 region, FLT3PMs in the pediatric cohort occurred in TKD domain (N=44), but commonly occurred in the transmembrane domain (TMD) and JMD. Twelve somatic mutations were identified at distinct positions within the JMD, the region involved in regulation of activity of the kinase. Among the JMD mutations, 9 occur at novel pediatric specific sites with significant activating potential (E573D/G, L576R, T582N, D586Y, Y589H, E596K/G, E598D, Y599C, D600G). Crystal structure analysis of FLT3 variants was used to assess the potential functional significance of the newly discovered variants. This structural modeling indicated that many of the mutations within the TMD (e.g. A680V) and TKD1 (L616R, M664I, M665L) were predicted to cause JMD destabilization, resulting in dysregulated activation of FLT3. Additionally, almost all JMD mutations have the potential to significantly disrupt the auto-inhibitory conformation, resulting in constitutive FLT3 activation. Mutations causing excessive activation of the kinase may have significant oncogenic capacity. In order to assess functional implications of the mutations, we cloned and expressed 6 of the most common novel variants (E573D, L576R, Y599C, D600G, F612L, and A680V) for functional assessment. Of the 6 mutations tested, 5 (E573D, L576R, Y599C, D600G, and A680V) resulted in auto-phosphorylation of FLT3, demonstrating dysregulated and enhanced kinase activation. Acquired mutations following tyrosine kinase inhibitor (TKI) exposure are heavily implicated in resistance and are almost exclusively confined to the two TKD regions, and the D835 position is a hotspot for resistance conferring mutations. It is important to identify patients at diagnosis who harbor dual mutations as this could indicate de novo resistance to TKIs, and exposure to these agents would have no efficacy, but may result in unnecessary toxicity. The presence of dual FLT3/ITD and FLT3/ALM mutations at diagnosis has been reported to occur at a very low prevalence. We analyzed the presence of co-occurring FLT3/ITDand FLT3 PMs in our pediatric cohort. Of the 128 patients with FLT3/ITD, 18 (14%) harbored a secondary FLT3 PM. Mutations in the JMD (N=8) and TMD (N=2) accounted for 56% of co-occurring mutations. Only 22% (N=4) mutations occurred at the D835/I836 sites, with an additional 22% (N=4) located at other TKD sites. Identified TKD diagnostic mutations included F612L, F616R, N676K/S, and N841K, a few which have only been reported in the setting of post TKI relapse. We identified novel pediatric specific FLT3 mutations with significant functional capacity. Importantly, these activating mutations may be uniquely susceptible to FLT3 inhibition and provide a therapeutic target in pediatric AML. Further work is ongoing to completely understand the oncogenic potential of each unique mutation and their prognostic and therapeutic implications. Disclosures No relevant conflicts of interest to declare.

Description: FLT3 is an receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of the differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with Acute myelogenous leukemia (AML) and confer poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations on the FLT3 kinase activation loop (e.g. D835V). Our laboratory is studying the signaling pathways associated with a newly identified multi-targeted tyrosine kinase receptor small molecule inhibitor (RTKI), ABT-869. Recently published work in our laboratory showed that using ABT-869 to treat MV4-11, a human AML FLT-3 ITD mutant cell line, resulted in the inhibition of phosphorylation of FLT-3 with a downstream inhibitory effect on the activation of STAT5, ERK, and Pim-1. Cell viability assays determined that MV-411 cells responded to ABT-869 in a concentration dependent manner (IC50 = 10nM). Apoptosis studies also showed an induction of apoptosis in ABT-869 treated cells. In vivo studies involving xenograft injections of MV-411 cells into SCID mice and subsequent treatment with ABT-869 demonstrated regression of tumor formation. In this study, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation is used as an isolated Flt-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells when compared to Ba/F3 Flt-3 D835V mutant and Ba/F3 Flt-3 WT cells. Trypan Blue Exclusion and Alamar Blue assays were used to demonstrate that there is 50% inhibition of growth and proliferation (IC50) of Ba/F3 FLT3 ITD mutant cells at a concentration of 1nM after 48 hours of treatment. Ba/F3 FLT3 D835V mutant cells show an IC50 between 1μM and 10μM after 48 hours of treatment. In contrast, Ba/F3 FLT3 WT cells demonstrate an IC50 of 10μM only after 72 hours of treatment. Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis (41.2%) occurred in Ba/F3 Flt-3 ITD mutant cells treated with 10nM ABT-869 after 24 hours when compared to untreated (6.5%) or vehicle control (6.1%) cells. Staining of Ba/F3 Flt-3 WT treated cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cell only and DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells following treatment with ABT-869 whereas no cleavage was observed with Ba/F3 WT cells treated with ABT-869. In vivo, the activity of ABT-869 treatment of SCID mice injected with Baf3 Flt-3 ITD, Baf3 Flt-3 D835V, or Baf3 Flt-3 WT cells is also being evaluated. Using bioluminescence imaging, it was determined that Ba/F3 FLT-3 ITD mutant and Ba/F3 Flt-3 D835Vmutant cell lines result in metastases and subsequent death in SCID mice after 2 weeks for ITD and 5 weeks for D835V, whereas mice injected with Ba/F3 WT survive longer than 5 weeks. Preliminary data demonstrated that ABT-869 prolonged survival in mice injected with the Ba/F3 FLT3-ITD cells compared to controls. Our preclinical data demonstrate that ABT-869 is effective specifically with FLT-3 ITD mutant cell lines in an isolated system. These studies provide rationale for the treatment of AML patients and the prevention of relapse.

Description: In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3–internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC50 approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC50 = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G0/G1 phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)–FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC50 approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC50 = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

Description: The Casitas B-Lineage Lymphoma (CBL) gene encodes for an E3 ubiquitin ligase that targets activated receptor tyrosine kinases for degradation. Mutations of the CBL gene have been described in juvenile myelomonocytic leukemia (JMML) but less is known about mutations and variants of CBL in de novo AML. We previously reported that somatic mutations of CBL are rare in pediatric AML. In this report we present a comprehensive evaluation of genomic and transcript variants of CBL including novel deletion events as well as transcript variants which, in combination with somatic mutations, account for over 6% of pediatric AML with extreme association with inv(16) and favorable outcome. Initial assessment of CBL transcript in a cohort of 100 patients identified previously reported deletion of exon 8 (CBL ΔE8, N=2) associated with CBL splice mutations as well as a novel whole exon 8 and 9 deletion variant (CBL ΔE8+9, N=3) without identifiable underlying somatic alterations. Long distance PCR, as well as custom Nanostring CNV array evaluation revealed a genomic deletion underlying this transcript variant. Subsequent whole genome sequencing as part of COG/NCI TARGET AML initiative, identified discrete genomic deletions of 1998, 3588 and 6189 bp across exon 8 and 9, leading to the generation of this novel variant. We evaluated the functional consequence of the novel CBL ΔE8+9 deletion variant by expressing it in IL3-dependent Ba/F3 cell line. Compared to control cells, Ba/F3 cells expressing CBL ΔE8+9 demonstrated cytokine independent growth. A comprehensive profiling of CBL variants was conducted in 796 pediatric de novo AML patients by transcript profiling (transcript variants) or by exome capture sequencing (somatic mutations including point mutations and smaller indels). All patients were treated on Children's Oncology Group studies AAML03P1 (N=167) and AAML0531 (N=629) and presence of CBL variants was correlated with disease characteristics and clinical outcome. Of the 796 patient specimens tested, 50 patients (6.3%) had one of 3 distinct CBL variants; transcript variant (N=28), somatic mutation (N=14), or dual transcript variant and somatic mutation (N=8). All cases of CBL ΔE8+9 were associated with a corresponding genomic deletion. Out of 14 cases of CBL ΔE8 and 1 case of CBL ΔE9, only 4 cases (27%) had a splice site mutation identified as the underlying mechanism of splice variant. Presence of CBL variants was correlated with clinical characteristics and outcome. Those with CBL variants had a significantly higher prevalence of inv(16) compared with CBL wild type (WT) (37% vs. 13%, p

Description: Activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) are present in ~80% of patients with Chronic Neutrophilic Leukemia (CNL) Despite the high frequency of these mutations in CNL, they are quite rare in adult acute myeloid leukemia (AML), in which only a single CSF3R mutated case was found in the TCGA AML analysis (0.5%). We have previously demonstrated significant variation in genomic variants between pediatric and adult malignancies, thus prevalence of genomic variants identified in adults need be fully characterized in children. As part of COG/NCI TARGET AML initiative we interrogated the genomic makeup of 200 cases of childhood AML (discovery cohort) using whole genome sequencing and performed subsequent frequency determination of the variants in 787 unselected cases from COG AAML0531 (validation cohort). Somatic variants in CSF3R were initially found to be recurrent in the discovery cohort and underwent frequency determination in the validation cohort to establish their prevalence and correlations with clinical characteristics and outcome. Frequency determination of CSF3R mutation in 787 pediatric patients with available CSF3R data from AAML0531 identified 16 distinct CSF3R mutations in 28 patients (3.6%). Somatic mutations in CSF3R identified in pediatric AML included known oncogenic variants mutations such as T618I and T615A, previously identified in adult CNL studies as well as novel truncations of the CSF3R cytoplasmic domain (Q749X, Y767fs, Y787X and P819/820fs), and missense mutations (E149D, A208V, R223Q, E405K, A431V, and Q516K). Interestingly, although CSF3R truncations usually occur along with a T618I or T615A mutation in CNL/aCML, these two mutation categories were mutually exclusive in pediatric AML. Initial correlation of all CSF3R variants with demographic and clinical/laboratory parameters determined that CSF3R variants were less prevalent in younger patients (age 0-2, p=0.039), with significantly higher association with t(8;21) (32% vs. 14%, p=0.012) and CEBPA mutations (35% vs. 5%, p

Description: Genetic alterations in the Nucleoporin (NUP) family of genes are involved in myeloid leukemogenesis and are associated with poor prognosis. We previously showed that NUP98-NSD1 is prevalent in acute myeloid leukemia (AML) and is highly associated with FLT3-ITD and dismal outcome. As genetic alterations in the NUP family are frequently cryptic by conventional karyotyping, their incidence has been underestimated. The COG/NCI TARGET AML initiative has performed comprehensive genome-wide characterization of diagnostic specimens from 200 pediatric AML cases in order to identify novel genetic lesions with prognostic and therapeutic significance. The interrogation of the whole genome and RNA sequencing data generated by this initiative identified numerous fusion transcripts involving the NUP family of genes, including NUP98-NSD1, NUP98-KDM5A, NUP98-HOXA9, NUP98-HMG3, NUP98-HOXD13, NUP98-PHF23, NUP98-BRWD3, CLINT-NUP98 and DEK-NUP214. All computationally identified NUP fusions were verified by orthogonal methodology and high-throughput screening assay was developed for frequency determination. The verified NUP fusions were screened in children treated on COG AAML0531 and AAML03P1 to define their prevalence, clinical characteristics and association with clinical outcome. The impact of NUP fusions was initially evaluated in patients with cytogenetically normal AML (CN-AML). NUP fusions were observed in 14.5% (35 of 242) patients: NUP98-NSD1 (N=21), DEK-NUP214 (N=3), NUP98-HMG3 (N=3), NUP98-HOXD13 (N=2), NUP98-PHF23 (N=2) and NUP98-KDM5A (N=4). The NUP fusions NUP98-BRWD3, NUP98-HOXA9 and CLINT-NUP98 were not found in CN-AML patients. Demographics and disease characteristics of CN-AML patients with and without NUP fusions were compared. Although patients of Asian descent comprised only 7% of the study population, they harbored significantly higher number of NUP fusions (29% vs 5%, P =0.002). Among those of Asian descent with CN-AML, 35% harbored a NUP fusion. We also noted an inverse association between NUP fusions and African-Americans where NUP fusions were not identified in any of African-American patients (P =0.031). NUP fusions were correlated with other common mutations in AML. NPM1 (9% vs 28%, P =0.007) and CEBPA (6% vs 19%, P =0.06) were rare in patients with NUP fusions, whereas FLT3/ITD (62% vs 34%, P =0.002) and WT1 (32% vs 8%, P

Description: Although survival of children with B-cell acute lymphoblastic leukemia (B-ALL) has improved substantially over time, 15% to 20% of patients will relapse, and most of those who experience a bone marrow relapse will die. A better understanding of genetic and epigenetic aberrations in relapsed ALL will facilitate new strategies for risk stratification and targeted therapy. In this collaborative study with the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project, we performed high resolution genome-wide DNA methylation profiling using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) array on a total of 178 (110 diagnosis, 68 relapse) leukemia samples from 111 patients with childhood B-ALL enrolled on the Children’s Oncology Group (COG) clinical trials who experienced relapsed, and 12 normal preB samples isolated from the bone marrows of 12 healthy individuals. The HELP array covers 117,521 CpG sites, annotated to ∼22,000 gene promoters. For eight diagnosis/relapse pairs, base-pair resolution DNA methylation using the eRRBS (enhanced Reduced Representation Bisulfite Sequencing) method was also performed on Illumina HiSeq2000. The median relapse time for the 111 patients was 21.8 months (range 2.1 to 56.2). Unsupervised clustering analysis using the HELP data revealed seven clusters: one cluster contained only the 12 normal preB samples; four clusters were enriched with MLLr, ETV6/RUNX1, Trisomy 4+10, and TCF3/PBX1 samples, respectively. The sixth cluster was not enriched for specific cytogenetic cases, but interestingly, all cases in this cluster were NCI High Risk (age〉10 years or WBC〉=50,000; p25%, FDR

Description: In efforts to discover genes uniquely expressed in childhood AML, we performed transcriptomesequencing (RNA-Seq) in pediatric AML and contrasted the expression signature to that in normal marrow hematopoiesis. This effort led to the discovery of over 200 genes that lack expression in normal hematopoietic cells, but are variably expressed in pediatric AML cells. Mesothelin(MSLN) was discovered to be one of the most highly expressed genes in a subset of childhood AML cases (p