ALBERT

Description: t(8;21) is the most frequent chromosomal abnormality in acute myeloid leukemia (AML), occurring in 4-12% of adult and 12-30% of pediatric patients. This translocation fuses the N-terminus of AML1 to nearly the entire coding region of ETO, resulting in expression of the fusion protein AML1-ETO. Observations that mice expressing AML1-ETO develop AML only if treated with mutagenic agents have suggested that AML1-ETO requires cooperating disease alleles for leukemogenesis. Consistent with this, t(8;21)+ AML patients harbor multiple genetic abnormalities. Recent exome/genome sequencing studies have expanded the number of known mutations in t(8;21)+ AML patients; however, efforts to distinguish driver from passenger mutations have yielded few cooperative events and the requirements for AML1-ETO leukemogenesis remain largely unknown. To better define the genetic landscape in AML and distinguish driver from passenger mutations, we compared the mutational profiles of two specific AML1-ETO driven mouse models of leukemia to the mutational profiles of human AML patients. We found that the mouse models of AML1-ETO driven AML were phenotypically similar in terms of their extensive latency, myeloid progenitor immunophenotype, and the acquired secondary disease alleles. The first model relies upon the expression of AML1-ETO in transplanted p21 null cells, while the second model relies upon the expression of AML1-ETO9a, a splice variant of AML1-ETO, in transplanted wild type cells. p21 is neither disrupted, nor methylated in t(8;21)+ AML. Because loss of p21 prevents the repair of damaged DNA, leukemogenesis may occur in this model once a cooperating disease allele has been naturally acquired in an AML1-ETO positive hematopoietic progenitor. AML1-ETO9a itself deregulates the expression of several DNA repair genes, suggesting that AML1-ETO9a could similarly facilitate the acquisition of a cooperating disease allele. When we compared the mutational landscape of these murine leukemias to AML patients, we found that the murine leukemias enrich for disease alleles present in human AML (hypergeometric p ≤ 4.26x10-20) and that there is a significant tendency for disease alleles mutated in both species to possess mutations in the same protein domain (hypergeometric p ≤ 4.23x10-3). Furthermore, domains mutated in both species were affected by recurrent mutations (Spearman correlation of domain p-values r = 0.53, p ≤ 2.73x10-8). While the frequency with which various protein classes were affected by mutations was significantly different in MLL-AF9 and AML1-ETO/AML1-ETO9a positive murine AML compared to MLL-fusion and t(8;21)+ positive human AML (p = 0.049), the protein classes targeted in AML1-ETO/AML1-ETO9a murine AML vs. human t(8;21)+ AML were not significantly different (p = 0.327). To identify disease alleles capable of cooperating with AML1-ETO, we determined that of the 424 genes mutated in both species, 38 of those genes were significantly mutated in human AML (Genome MuSiC SMG FDR ≤ 30%). These 38 genes represented 45 mouse orthologues, 38 of which were significantly mutated in AML1-ETO driven murine leukemias (FDR ≤ 10%). These 38 orthologues corresponded to 32 human orthologues, 3 of which were annotated in COSMIC as cancer-related genes: TET2, PTPN11, and THRAP3. Using retroviral transduction and transplantation experiments, we demonstrated that the expression of AML1-ETO in transplanted Tet2 null cells or PTPN11 D61Y cells was sufficient for leukemogenesis. At euthanasia, mice exhibited leukocytosis, anemia, thrombocytopenia, splenomegaly, and an expansion in the myeloid progenitor compartment. Our identification of Tet2 loss as a cooperating allele implicates mutations in epigenetic regulators as potential driving events in t(8;21)+ AML, while the discovery of PTPN11 D61Y solidifies the role of constitutive MAPK signaling in t(8;21)+ AML. This integrative genetic profiling approach allowed us to accurately predict cooperating events in t(8;21)+ AML in a robust and unbiased manner, while also revealing functional convergence in mouse and human AML. Collectively, these findings illustrate the power of integrating murine and human genomic profiling to identify functionally relevant disease alleles in AML. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

Description: Abstract 308 Loss-of-function somatic mutations in Addition of Sex Combs Like 1 (ASXL1) occur in a subset of patients with myeloid malignancies, most commonly in myelodysplastic syndrome (MDS). In addition, germline mutations in ASXL1 are observed in patients with Bohring-Opitz Syndrome, a developmental disorder characterized by neurologic and skeletal abnormalities. Previous work characterizing a constitutive gene-trap Asxl1 model was notable for significant perinatal lethality of Asxl1−/− mice with evidence of an overt hematopoietic phenotype in surviving mice. Given the lack of detailed data on the hematopoietic phenotype of Asxl1 loss in vivo, we created a murine model for conditional knockout of Asxl1 for tissue- and temporal-specific deletion with 4 different Cre recombinase alleles. We crossed our conditional Asxl1 allele to EIIa-cre to generate mice with germline loss of Asxl1; this revealed embryonic lethality of mice with homozygous germline Asxl1 loss. Mice with heterozygous germline deletion of Asxl1 were viable and fertile although a significant proportion had cranio-facial abnormalities. Timed-sacrifice of pregnant mothers from heterozygous EIIa-cre Asxl1+/− crosses revealed that homozygous Asxl1−/− pups survived to 18–20.5 days post-coitus, with all embryos characterized by craniofacial abnormalities including anopthalmia, microcephaly, cleft palates, and mandibular malformations similar to those seen with Bohring-Opitz syndrome. We then generated mice with conditional Asxl1 deletion in the hematopoietic compartment by crossing floxed mice with Vav-cre recombinase mice for hematopoietic deletion at birth, and with Mx1-cre mice for inducible deletion of Asxl1. Deletion of Asxl1 in both systems resulted in complete loss of Asxl1 as assessed by Western blot. In both models we noted progressive development of leukopenia and anemia in mice with homozygous loss of Asxl1 compared to age-matched controls (Figure). This was associated with extramedullary hematopoiesis and morphologic evidence of myeloid, erythroid, and megakaryocytic dysplasia, similar to that observed in human MDS (Figure). Flow cytometry revealed a progressive increase in immunophenotypically-defined multipotent progenitors (Lineage-negative, Sca1+, cKIT+, CD150-, CD48+ cells) in bone marrow and spleen of knockout (KO) mice. Characterization of HSC's from 6-week old Vav-cre Asxl1−/− mice using serial competitive transplantation revealed a competitive disadvantage with transplantation of SLAM+ cells from 6-week old KO mice. A subset of Vav-cre Asxl1−/− mice developed a transplantable, monocytic-like leukemia beyond 6 months of age which was characterized by both proliferative and dysplastic features. Restriction of Asxl1 deletion to the megakaryocytic compartment using Pf4-cre revealed an age-specific decline in platelet production in Pf4-cre Asxl1−/− mice compared to age-matched controls, and a concomitant increase in bone marrow megakaryocytes in KO mice. Consistent with previous in vitro studies, hematopoietic-specific deletion of Asxl1 was associated with a marked decrease in H3K27me3 as assessed by histone Western blots of murine splenocytes and H3K27me3 ChIP-Seq of myeloid progenitors from 1 year-old Vav-cre Asxl1−/− mice compared with littermate controls. RNA-Seq from myeloid progenitors was integrated with H3K4me3/K27me3 ChIP-Seq to identify gene targets of Asxl1 loss associated with myelodysplasia. Given that MDS patients frequently present with concomitant ASXL1 and TET2 mutations, Vav-cre Asxl1fl/fl mice were also crossed with mice bearing floxed alleles of Tet2. Mice with combined hematopoietic-specific deletion of both Asxl1 and Tet2 developed bone marrow failure and hastened death compared with age-matched, single-gene deleted counterparts at 25 to 40 weeks of age. The findings here reveal that hematopoietic-specific deletion of Asxl1 results in progressive ineffective hematopoiesis, an increase in hematopoietic progenitors, a propensity for leukemic transformation with age, and morphologic features of human MDS (Figure). Combining Asxl1 deletion with loss of Tet2, a combined genotype present in at least 5% of patients with de novo MDS, resulted in shorter latency, progressive MDS. These data suggest that these two models represent novel, genetically accurate models of MDS amenable to epigenomic, functional and preclinical therapeutic studies. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Aid loss leads to altered differentiation, transcription, and methylation in specific genetic loci in hematopoietic stem/progenitor cells. Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD in myeloid leukemogenesis.

Description: Abstract 405 Somatic mutations in ASXL1 have been identified in patients with myeloid malignancies and are associated with worsened overall survival in AML and MDS patients. However the mechanisms of myeloid transformation of ASXL1 mutations had not been delineated. We therefore performed extensive in vitro and in vivo studies to assess the functional implications of ASXL1 mutations in the hematopoietic compartment. Transcriptional and Western blot analysis demonstrated loss of ASXL1 protein in primary leukemia samples with endogenous ASXL1 mutations indicating that these mutations are loss-of-function disease alleles. Further, ASXL1 depletion by shRNA in normal and malignant hematopoietic cells leads to robust upregulation of a set of genes including the posterior HOXA cluster (HoxA5-HoxA13). Increased HoxA gene expression was confirmed in human hematopoietic stem progenitor cells targeted with ASXL1 siRNA and in mice with conditional deletion of Asxl1 in the hematopoietic compartment. Previous studies in Drosophila had revealed that Asxl forms the polycomb-repressive deubiquitinase (PR-DUB) complex with BAP1, which normally opposes the function of polycomb repressive complex 1 (PRC1) by removing H2AK119 ubiquitination. We verified that wild-type, but not mutant ASXL1 associates with BAP1 in co-immunoprecipitation studies. However, BAP1 depletion in hematopoietic cells did not result in significant changes in HoxA gene expression, suggesting that ASXL1 regulates gene expression in hematopoietic cells independent of its role in the PR-DUB complex. We therefore performed CHIP sequencing for known activating and repressive chromatin marks and histone mass spectrometry to elucidate the genome-wide effects of ASXL1 loss on chromatin state in hematopoietic cells. This allowed us to show that ASXL1 loss resulted in genome-wide loss of the transcriptionally repressive mark H3K27me3 in hematopoietic cells and primary patient samples with ASXL1 mutations. These data were supported by western blot analysis and histone mass spectrometry demonstrating a significant loss of H3K27 trimethylation in ASXL1-mutant cells. Moreover, ASXL1 mutations in primary leukemia samples are characterized by loss of H3K27 trimethylation at the HoxA locus. These data led us to hypothesize that ASXL1 interacts with the PRC2 complex; co-immunoprecipitation studies revealed that ASXL1 associates with members of the PRC2 complex including EZH2 and SUZ12 but not with the PRC1 repressive complex. Importantly, ASXL1 downregulation resulted in loss of EZH2 recruitment to the HOXA locus indicating a role of ASXL1 in recruiting the PRC2 complex to known leukemogenic loci. We next assessed the effects of ASXL1 loss in vivo by generating a conditional knock-out model of ASXL1 and also by employing shRNA to deplete ASXL1 in hematopoietic cells expressing the NRASG12D oncogene. Consonant with the in vitro data, we observed HOXA9 overexpression with ASXL1 loss/depletion in vivo. Preliminary analysis reveals that conditional, hematopoietic specific ASXL1-knockout (ASXL1fl/fl Vav-Cre) mice are characterized by progressive expansion of LSK and myeloid progenitor cells in mice less than 6 months of age. After 6 months of age a significant proportion of ASXL1fl/fl Vav-Cre mice developed leukocytosis, anemia, thrombocytopenia, and splenomegaly; pathologic analysis of tissues revealed a phenotype consistent with myelodysplasia with myeloproliferative features. Moreover, loss of ASXL1 in cooperation with expression of NRasG12D resulted in impaired survival, increased myeloproliferation, and progressive anemia consistent with MPN/MDS in vivo. Taken together, these results reveal that ASXL1 mutations result in a loss-of-function and suggest a specific role for ASXL1 in epigenetic regulation of gene expression by facilitating PRC2-mediated transcriptional repression of known leukemic oncogenes. Moreover, our in vivo data validate the importance of ASXL1 mutations in the pathogenesis of myeloid malignancies and provide insight into how mutations that inhibit PRC2 function contribute to myeloid transformation through epigenetic dysregulation of specific target genes. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

Description: Abstract 2802 Background: Therapy-related myelodysplastic syndromes and acute myelogenous leukemia (tMDS/AML) comprise a poor-risk subset of MDS/AML and are associated with a higher rate of cytogenetic abnormalities and complex karyotypes. Large scale mutation profiling efforts in de novo MDS have identified mutations that correlate with clinical features but such mutations have not been investigated in tMDS/AML. Methods: Cryopreserved (mononuclear cell fractions) bone marrow and peripheral blood samples from tMDS/AML patients were analyzed. Lymphocytes were depleted from these fractions by either fluorescence-activated cell sorting or affinity column selection. Genomic DNA was subjected to high throughput PCR and sequenced for TP53, TET2, DNMT3a, ASXL1, IDH1, IDH2, SF3B1, EZH2, EED, SUZ12, and RBBP4. Somatic mutations were validated by comparison to lymphocyte DNA controls. Patient selection was based on sample availability for tMDS/AML patients with untreated or active, previously treated disease. Results were correlated with clinical outcomes, cytogenetic profiles, and response to therapy. Results: Samples from 38 patients (20 males, 18 females) with tMDS/AML were analyzed. For their primary malignancy (≥ 2 malignancies, n=3; AML, n=1; breast, n=4; colorectal, n=3; Hodgkins or composite lymphoma, n=3; gastric, n=1; melanoma, n=2; NHL, n=12; ovarian, n=1; prostate, n=3; sarcoma, n=2; thyroid, n=3), patients received chemotherapy alone (n=17), radiation alone (n=4), radioactive iodine alone (n=1), chemotherapy plus radiotherapy (n=13), radiotherapy plus radioactive iodine (n=1), chemotherapy plus radiotherapy plus radioactive iodine (n=2). Median latency time between primary malignancy treatment and MDS diagnosis date was 5.68yrs (range, 0.71–30.88). Median age at MDS diagnosis was 65yrs (range, 34–83). Not surprising was the finding that of the 35 MDS patients with complete IPSS parameter data most were IPSS Int-2 (45%) or High Risk (13%) compared to Low (11%) or Int-1 Risk (24%) disease, and in contrast to expected proportions at diagnosis for de novo MDS. WHO Classifications were: RA, n=3; RCMD, n=11; RAEB-1, n=7; RAEB-2, n=7; AML, n=3; CMML-1, n=1; MDS-U, n=3; Unknown, n=3. The median survival was 16.8mo. Median time between MDS diagnosis and sample procurement was 3.0mo (range, 0–57.2) during which 2 patients progressed to AML, 11 received a DNA methyltransferase inhibitor (DNMTI), 1 induction chemotherapy, and 2 DNMTI plus induction. We identified somatic mutations in 15/38 (39.5%) patients. Including cytogenetic abnormalities, we identified somatic alterations in 34/38 (88%) of the patients in this cohort. TP53 mutations were most common, detected in 8/38 (21%) patients, followed by TET2 in 4/38 (10.5%), DNMT3a in 3/38 (7.9%), ASXL1 in 1/38 (2.6%), IDH1 in 1/38 (2.6%), and EZH2 in 1/38 (2.6%). No IDH2, SF3B1, EED, SUZ12, or RBBP4 mutations were detected. Only 2 patients had concurrent point mutations (one patient with TP53/TET2/DNMT3 mutations and one patient with TET2/EZH2 mutations). 7/38 (18.4%) patients had TP53 loss by FISH analysis or exhibited loss of chromosome 17/17p; 2 of these patients showed concurrent TP53 point mutations consistent with biallelic TP53 loss. 12/13 patients with TP53 abnormalities (point mutations, loss of TP53 by FISH, or loss of chromosome 17) had IPSS Poor Risk cytogenetics and 11/13 patients had abnormalities in chromosome 5 (del 5q or monosomy 5). In patients without TP53 abnormalities, 9/25 had Poor Risk cytogenetics and 5/25 had abnormalities in chromosome 5. The median survival for patients with TP53 abnormalities was 9.7mo compared to 64.4mo for patients with no TP53 abnormalities (p=0.0043). Of the 13 patients with TP53 abnormalities, 12 received treatment for tMDS/AML. 3/12 were refractory to the first line of therapy, and 7 were unable to receive an adequate course of therapy with hypomethylating agents due to toxicity or progressive disease. Conclusions: TP53 point mutations are more common in tMDS/AML than in de novo MDS (7.5%, Bejar et al) while incidence of TET2 and ASXL1 mutations were lower in tMDS/AML compared to de novo MDS (20.5% and 14.4%, respectively). TP53 and TET2 point mutations were more strongly associated with exposure to prior chemotherapy, but not with exposure to radiation therapy. Taken together, these data demonstrate that TP53 mutations are common in tMDS/AML and are correlated with adverse clinical outcomes. Disclosures: No relevant conflicts of interest to declare.

Description: Epigenetic modifiers and signaling factors are frequently mutated and often co-occur in various myeloid malignancies. However, precisely how these mutations cooperate to cause myeloid leukemia is not fully understood. Here, we show that cells with concurrent Ten-eleven-translocation 2 (Tet2) loss and Nras mutation can cause lethal chronic myelomonocytic leukemia (CMML) like disease in vivo and synergistically activate Ras signaling through epigenetic silencing of Sprouty2 (Spry2), thereby making cells with both disease alleles dependent on MAPK signaling and highly sensitive to MEK inhibition. To assess if Tet2 loss and Nras mutation cooperate in myeloid transformation, we crossed Tet2 conditional knockout mice (Mx1-Cre+Tet2f/f) and Nras mutant mice (Mx1-Cre+Nras+/G12D) to generate Mx1-Cre+Tet2f/fNras+/G12D mice (Tet2Δ/ΔNras+/G12D). These mice, compared to single mutant mice with either allele alone, had more significant monocytosis, expansion of Lineage- Sca-1+ c-Kit+ (LSK) and myeloid progenitors in both bone marrow (BM) and spleen and development of lethal CMML-like disease (median survival 264 days). Moreover, serial transplantation of splenic cells derived from leukemic Tet2Δ/ΔNras+/G12D mice caused similar CMML-like disease in recipients, which emanates from LSK-positive stem/progenitor cells as the disease propagating population. To delineate how Tet2 loss and Nras mutation synergize in leukemic transformation, we next performed western blot and phospho-flow analysis of MAPK and PI3K signaling in primary hematopoietic cells. Interestingly, pErk, pAkt and pS6 expression were significantly higher in Tet2Δ/ΔNras+/G12D cells compared to WT or single mutant cells, indicating that Tet2 loss and Nras mutation cooperates to further activate Ras signaling (Figure 1). Consistent with our murine model, TET2 silencing in NRAS mutant human leukemia cells increased MAPK output, consistent with augmentation of signaling by concurrent TET2/NRAS alterations in human leukemia cells. To unravel the molecular mechanism of Ras signaling activation, we assessed mRNA / protein expression and performed bisulfite sequencing of known regulators of MAPK signaling, including Sprouty family members. We observed significant decrease of Spry2 expression, stepwise and specific hyper-methylation of CpG islands in the Spry2 promoter region in Tet2Δ/ΔNras+/G12D cells compared to WT or single mutant cells, consistent with progressive epigenetic remodeling in these leukemia cells in vivo (Figure 2). Genome wide methylation profiling of WT, single mutant and Tet2Δ/ΔNras+/G12D LSK cells using enhanced reduced representation bisulfite sequencing demonstrated clear separation of leukemic Tet2Δ/ΔNras+/G12D LSKs from WT or single mutant LSKs. Most importantly, restoration of Spry2 expression in Tet2Δ/ΔNras+/G12D cells led to decrease in pErk / pAkt level and significantly reduced colony formation, which functionally validates Spry2 as a key epigenetic target in Tet2/Nras mutant leukemia cells. We next assessed whether the increased MAPK signaling seen in Tet2Δ/ΔNras+/G12D cells leads to differential sensitivity to MEK inhibition by performing studies with the clinical MEK inhibitor binimetinib (ARRY162). Tet2Δ/ΔNras+/G12D cells showed significantly higher sensitivity to binimetinib compared to Nras+/G12D cells in vitro (IC50, 6.948nM vs. 690.4nM). Moreover, in vivo treatment of Tet2Δ/ΔNras+/G12D leukemic recipients with binimetinib restored splenomegaly, significantly reduced disease burden in BM and spleen and improved overall survival compared to vehicle treatment (median survival 24.5 days vs. 44.5 days, p=0.0018, Figure 3). Of note, knockdown of human TET2 in NRAS mutant human leukemia cells sensitized to MEK inhibition in a similar manner demonstrating this approach may have value in leukemia patients with concurrent TET2 and NRAS mutations. These data clearly indicate that Tet2 loss and Nras mutation synergize in myeloid transformation and cooperatively remodel DNA methylation, which leads to epigenetic silencing of Spry2 and synergistic activation of MAPK signaling, which can be leveraged through therapeutic MEK inhibition. Our studies provide novel insights into how signaling and epigenetic mutations cooperate in leukemic transformation and provide a rationale for mechanism based therapy in CMML patients with these high risk genetic lesions. Disclosures Melnick: Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Description: Therapy-related Myeloid Neoplasms (tMN) comprise a poor risk subset of myelodysplastic syndromes and acute myelogenous leukemia, are increasing in incidence, and represent a serious complication following treatment for primary malignancies. In our previous study of 11 genes in 38 tMN patient samples, the data suggested that the mutational spectrum of tMN was distinct from de novo myeloid malignancies. To confirm this finding and to refine the tMN mutation profile, we investigated the mutation profile in samples from 88 patients and 28 genes using Sanger and next-generation sequencing approaches. We performed amplification using RainDance microfluidic PCR, followed by HiSeq next-generation sequencing. Mutations were identified using a modified pipeline for SNP calling employing variant detection software programs. Our study cohort included 88 patients, 71 of whom had complete clinical data for analyses. Patients had a history of epithelial and hematologic malignancies (³2 malignancies n=11; breast n=9; colorectal n=5; head and neck n=4; genital-urinary n=6; lung n=1; lymphoma n=25; melanoma n=2; ovarian n=1; sarcoma n=2; other, n=5). Treatment of primary cancers included chemotherapy alone (n=27), radiation alone (n=8), autologous stem cell transplant (n=11), or chemotherapy plus radiation (n=25). The median latency time between primary malignancy treatment and tMN diagnosis was 5.7yrs (range, 0.7 - 30.9 yrs). Median age at tMN diagnosis was 64yrs (range, 26 - 85 yrs). International Prognostic Scoring System (IPSS) risk group for MDS at tMN diagnosis were Low risk (n=8), Int-1 (n=11), Int-2 (n=30), High risk (n=9). We identified somatic mutations in 56 of 88 (64%) patients (83 patients were evaluated by next-generation sequencing and 5 by Sanger sequencing only). Mutations in TP53 were most common and were detected in 27/88 patients (30.7%), followed by mutations in TET2 in 12/88 (13.6%), DNMT3A in 9/88 (10.2%), NRAS in 8/83 (9.6%), KRAS in 5/83 (6.0%), and KIT in 5/83 (6.0%). Gene mutations detected at lower frequencies included those in ASXL1 in 5/88 (5.7%), RUNX1 in 2/83 (2.4%), EZH2 in 1/88 (1.1%), and SF3B1 in 1/88 (1.1%). Of the 58 patients with complete sequencing and FISH data, 4 patients exhibited biallelic somatic TP53 mutations and 3 patients had TP53 mutation combined with del 17p TP53 loss, demonstrating that 7 of 58 evaluable patients (12.1%) experienced biallelic loss of TP53. We also identified biallelic mutations in TET2 and DNMT3A in 2 separate patients. 25 patients had 2 or more concurrent somatic mutations. The highest number of co-occurring mutations in one patient was 5 mutations; 12 patients had 2 somatic mutations. The most common co-occurrence was TP53 and TET2, which was observed in 5 patients. All 5 ASXL1 mutations co-occurred with additional mutations. By analyzing variant allele frequencies (VAFs) in patients with multiple mutations, we observed that some tMN patients harbored multiple clones with distinct VAFs. This observation was also supported by the co-occurrence of typical class I driver mutations in the same patient, (e.g. KRAS 6% and NRAS 21% VAF; NRAS 9% and KIT 34%; NRAS 26% and KIT 9% in individual patients). The allele frequency data also suggested that ASXL1 is likely an early occurring mutation as the VAF was higher than for other co-occurring mutations (mean VAF ASXL1 50%, other co-occurring genes 23.5%, p

Description: Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) cells, but not GMP cells, were able to induce disease in secondary and tertiary recipients in vivo. In order to assess the sensitivity of Tet2/Flt3-mutant AML and specifically the LSCs, to targeted therapies, we treated primary and transplanted mice with chronic administration of AC220, a FLT3 inhibitor in late-stage clinical trials. AC220 treatment inhibited FLT3 signaling in vivo, and reduced peripheral blood counts/splenomegaly. However, FLT3 inhibition did not reduce the proportion of AML cells in the bone marrow and peripheral blood. AC220 therapy in vivo reduced the proportion of GMP cells, but not LSK cells, demonstrating LSCs in this model are resistant to FLT3-targeted anti-leukemic therapy. We hypothesized that Tet2/Flt3-mutant LSCs possess a distinct epigenetic/transcriptional signature that contributes to leukemic cell self-renewal and therapeutic resistance. We performed RNA-seq using the Lifetech Proton sequencer to profile the expression landscape of Vav+Tet2fl/flFlt3-ITD mutant LSKs compared to normal stem cells. We were able to obtain an average of 62 million reads per sample. We identified over 400 genes differentially expressed in LSCs relative to normal hematopoietic stem cells (FC〉2.5, padj