AML1-ETO9a Research Articles

Novel Role of DHX15 in Modulating AML/ETO9a Spliced Variant Production in AML/ETO + AML

AML1/ETO9a(AE9a) is an alternatively spliced isoform of AML1/ETO(AE) fusion protein that coexists with AE and predicts a poor prognosis in AE-positive (AE +) AML. The insert of exon9a between exon8 and exon9 of ETO provides a stop codon forming the truncated AE9a lacks ETO domains NHR3/4, and the variant alone can induce leukemia. To date, its production mechanism remains obscure. Our previous studies showed that the splicing factor DHX15 is highly expressed in AML and correlates with poor prognosis (PMID: 29163777). DHX15 is also related to alternative splicing events in AE + AML revealed in an in vitro study (PMID: 27798625). Thus, we further investigated the potential role of DHX15 in AE + AML progression and AE9a splice variant production. To figure out the DHX15 expression level in AE + AML, bone marrow (BM) cells from 114 AE - AML (Fujian Union Hospital, year 2011-2018) and 104 AE + AML (part from Fujian Union Hospital, n=21, part from Wuhan Kindstar Diagnostics Co., Ltd, n=83, year 2016-2017) were collected. The RT-qPCR results showed that the expression of DHX15 in AE +AML is lower than those in AE - AML (P<0.0001), which is in line with the previous studies showing that a better prognosis is observed in AE + and DHX15-low expressed AML. The AE9a/AE ratio and DHX15 mRNA level in 98 AE + AML BM samples were further analyzed. The results showed that the AE9a(P<0.0002) and AE9a/AE ratio(P<0.0001) were significantly higher in DHX15 low expression AE + AML group (groups were divided based on medium expression level). Conversely, the DHX15 expression level was higher in AE9a/AE low group(P<0.0001), indicating a strong negative correlation between the mRNA level of DHX15 and AE9a/AE ratio in AE + AML. To further investigate how DHX15 impacts the spliced variant AE9a expression, DHX15 was knockdown (KD) in AE + AML cells. The AE9a/AE ratio was increased (P=0.0013) in DHX15-KD group compared to the control group. Gene set enrichment analysis (GSEA) of RNA-seq data revealed suppressed splicing and splicing complex assembly pathways in DHX15-KD Kasumi-1/SKNO-1 cells. These data demonstrated that downregulation of DHX15 increased AE9a expression probably through aberrant alternatively splicing process. RNA immunoprecipitation (RIP) assay showed AE and AE9a mRNA were detected in anti-DHX15 immunoprecipitants. However, DHX15 protein was not exclusively detected in AE9a-RNA pulldown-based Western blot (WB) or mass spectrometry (MS), suggesting DHX15 may not directly interact with AE or AE9a mRNA. To unravel the association between DHX15 protein and AE9a mRNA, AE and AE9a full-length mRNA pulldown-based MS were performed. We found that AE expression may rely more on spliceosome E complex, while AE9a splicing may rely more on spliceosome A complex. Moreover, the hnRNP protein HNRNPL and spliceosome A complex members SF3A2 and SRSF4 are exclusively combined with exon9a. Clinically, HNRNPL and SF3A2 were highly positively correlated with AE9a and DHX15 revealed in the Pearson analysis on mRNA expression of 16 AE + AML BM samples. Moreover, DHX15 may interact with HNRNPL and SF3A2, which were analyzed by protein-protein interaction analysis from Pathway Commons Protein-Protein Interactions and String. Collectively, DHX15 may modulate AE mRNA splicing together with HNRNPL and SF3A2, and its downregulation causes increased AE9a spliced variant production in AE + AML. In addition, at the protein level, we found the RecA1 domain of DHX15 can directly interact with the AE fragments containing RHD and NHR4 domains shown in immunofluorescence(IF) and co-immunoprecipitation (CO-IP) assays in vitro, which may contribute to the full-length production of AE. Moreover, DHX15 knockdown significantly decreased cell proliferation and cell cycle arrest through ATM/AKT-p53-p21 pathway in AE-positive AML cell lines demonstrated by cell proliferate, GSEA, and immunoblotting assays. These results suggest that DHX15 also plays a unique role in AML/ETO + AML oncogenesis. In conclusion, our studies focused on the AE + AML and revealed DHX15 regulates AE9a expression by alternative splicing together with HNRNPL and SF3A2. Since AE-positive AML is a heterogeneous disease with different phenotypes and varied prognoses based on expression patterns of the other four genes, our study provides new insights into DHX15 upon AML1/ETO9a splice variant production and DHX15 might be served as a therapeutic target in AE9a + AML.

Single-cell RNA sequencing of a new transgenic t(8;21) preleukemia mouse model reveals regulatory networks promoting leukemic transformation.

T(8;21)(q22;q22), which generates the AML1-ETO fusion oncoprotein, is a common chromosomal abnormality in acute myeloid leukemia (AML) patients. Despite having favorable prognosis, 40% of patients will relapse, highlighting the need for innovative models and application of the newest technologies to study t(8;21) leukemogenesis. Currently, available AML1-ETO mouse models have limited utility for studying the pre-leukemic stage because AML1-ETO produces mild hematopoietic phenotypes and no leukemic transformation. Conversely, overexpression of a truncated variant, AML1-ETO9a (AE9a), promotes fully penetrant leukemia and is too potent for studying pre-leukemic changes. To overcome these limitations, we devised a germline-transmitted Rosa26 locus AE9a knock-in mouse model that moderately overexpressed AE9a and developed leukemia with long latency and low penetrance. We observed pre-leukemic alterations in AE9a mice, including skewing of progenitors towards granulocyte/monocyte lineages and replating of stem and progenitor cells. Next, we performed single-cell RNA sequencing to identify specific cell populations that contribute to these pre-leukemic phenotypes. We discovered a subset of common myeloid progenitors that have heightened granulocyte/monocyte bias in AE9a mice. We also observed dysregulation of key hematopoietic transcription factor target gene networks, blocking cellular differentiation. Finally, we identified Sox4 activation as a potential contributor to stem cell self-renewal during the pre-leukemic stage.

Wnt and Notch Pathways Regulate Stemness of Myeloid Leukemia Cells Via IL33/ST2 Signaling

Therapy-resistant leukemic stem cells (LSCs) are a main cause of relapse in chronic and acute myeloid leukemia (CML and AML) patients. Stemness, a cellular state of LSCs characterized by their quiescence, pluripotency, and long-term self-renewal capacity, is thought to have major implications on therapy resistance and outcome in myeloid leukemias. However, signaling pathways that induce stemness are often not specific for LSCs and shared with normal hematopoietic stem cells (HSCs). Therefore, LSCs are difficult to be targeted therapeutically. IL-33/ST2 signaling may be a potential target to eradicate CML and AML LSCs. To investigate the ST2 gene (IL1RL1) expression in different hematological malignancies, we used the MILE gene expression dataset containing 2096 samples. The IL1RL1 high expressing samples (n=62) were exclusively represented by myeloid leukemias (CML and AML), and 50% of the IL1RL1 high samples harbored either the BCR/ABL1 (CML) or the AML1/ETO (AML) oncogenes. We confirmed the result from the MILE database by analyzing the ST2 protein expression on FACS-sorted CD34+ LSCs from AML and CML patient samples. Indeed, AML1/ETO and BCR/ABL1 harboring LSCs expressed ST2, while healthy hematopoietic stem cells (HSCs) and LSCs harboring other oncogenes were ST2-negative. Transfecting the ST2-negative cell line Ramos with the AML1/ETO and BCR/ABL1 oncogenes induced ST2-expression. Next, we performed a gene set enrichment analysis (GSEA), comparing the IL1RL1 high vs. IL1RL1 low samples in the MILE dataset. Genes involved in stemness (p<0.01), Wnt- (p<0.001), and NF-ΚB (p=0.054) signaling were upregulated, while the Notch-pathway (p<0.05) was downregulated. Since the NF-ΚB is a known downstream pathway of ST2, we hypothesized that Wnt and/or Notch signaling pathways regulate ST2 expression. Therefore, we treated FACS-sorted CD34+ AML1/ETO expressing LSCs with the Wnt pathway inhibitor XAV939 or HES1 and NOTCH1 shRNAs, silencing the Notch pathway. Treatment with XAV939 led to a decrease in ST2 expression in these cells, while silencing HES1 and NOTCH1 enhanced ST2-expression. To analyze the molecular mechanisms induced by IL-33/ST2 signaling, we performed a transcriptomic analysis using FACS-sorted CD34+ AML1/ETO LSCs upon treatment with or without IL-33. In total, 377 genes were up- and 139 genes downregulated after IL-33 stimulation. IL-33-induced genes were involved in the positive regulation of NF-ΚB (p<0.05) and canonical Wnt (p<0.001) signaling pathways, and the regulation of stem cell division and maintenance (p<0.001). To functionally test whether IL-33/ST2 signaling triggers stemness in myeloid leukemia, we performed a colony forming assay using CD34+ AML1/ETO or BCR/ABL1 expressing LSCs from leukemia patient samples. The colony numbers increased 1.5-3fold upon IL-33 treatment in three consecutive platings, indicating an increase in stem cell numbers. IL-33 had no effect on colony numbers of HSCs and LSCs harboring other oncogenes. To test the in vivo relevance of IL-33/ST2 signaling in leukemia development, we used a congenic AML1/ETO9a (AE9a) or BCR/ABL1 (CML) mouse model. Briefly, we transduced ST2-negative HSCs originating from BL/6 or ST2-knockout (ST2-/-) mice with AE9a-GFP or BCR/ABL1-GFP and transplanted those cells into recipients. In both models, the LSCs strongly expressed ST2, but IL-33/ST2 signaling had no influence on the disease development. Stemness supports resistance against standard therapy in both, AML and CML. Since we were able to show that IL-33/ST2 signaling promotes stemness in AML1/ETO and BCR/ABL1 LSCs, we hypothesized that IL-33 addition protects LSCs from treatment with standard therapeutics. Indeed, IL-33 rescued the reduced colony forming capacity of LSCs that were either treated with the chemotherapeutic agent cytarabine (AML1/ETO LSCs) or the tyrosine kinase inhibitor Nilotinib (BCR/ABL1 LSCs). Furthermore, ST2-deficiency led to a reduced LSC count in CML mice compared to control mice when treated with Nilotinib (p=0.047). We showed that ST2 is expressed on AML1/ETO and BCR/ABL1 harboring LSCs and that activated IL-33/ST2 signaling induced stemness in these LSCs. Targeting IL-33/ST2 signaling in these specific subtypes of AML and CML may eliminate LSCs and overcome resistance towards chemotherapy or TKIs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Reinforced erythroid differentiation inhibits leukemogenic potential of t(8;21) leukemia.

Oncoprotein AML1-ETO (AE) derived from t(8;21)(q22;q22) translocation is typically present in a portion of French-American-British-M2 subtype of acute myeloid leukemia (AML). Although these patients have relatively favorable prognoses, substantial numbers of them would relapse after conventional therapy. Here, we explored whether reinforcing the endogenous differentiation potential of t(8;21) AML cells would diminish the associated malignancy. In doing so, we noticed an expansion of immature erythroid blasts featured in both AML1-ETO9a (AE9a) and AE plus c-KIT (N822K) (AK) murine leukemic models. Interestingly, in the AE9a murine model, a spontaneous step-wise erythroid differentiation path, as characterized by the differential expression of CD43/c-Kit and the upregulation of several key erythroid transcription factors (TFs), accompanied the decline or loss of leukemia-initiating potential. Notably, overexpression of one of the key erythroid TFs, Ldb1, potently disrupted the repopulation of AE9a leukemic cells in vivo, suggesting a new promising intervention strategy of t(8;21) AML through enforcing their erythroid differentiation.

Leukemia Stem Cells Depend on Regulated Protein Synthesis

Acute myeloid leukemia (AML) is initiated and sustained by leukemia stem cells (LSCs) which arise from progenitor cells that do not usually self-renew but become aberrantly self-renewing. It is thought that LSCs gain aberrant self-renewal potential by co-opting molecular and cellular programs from hematopoietic stem cells (HSCs) (PMID: 16862118). HSCs have been shown to require tightly regulated protein synthesis rates, where increased or decreased protein synthesis impairs self-renewal (PMID: 24670665), but it is not known if LSCs share this dependence. We have shown that human LSCs reside in the population of AML cells with the highest levels of CD99 (PMID: 28123069). In RNA-sequencing studies, we found that LSCs with high levels of CD99 are depleted for ribosomal protein transcripts. We thus reasoned that similar to HSCs, LSCs may depend on tightly regulated protein synthesis to self-renew.To test if CD99 promotes LSC function by constraining protein synthesis, we transduced c-Kit+ cells from B6-CD99 Gt(pU-21T)44lmeg (CD99 KO) or wild-type (WT) mice to express AML1-ETO9a (AE9a) and transplanted them into WT mice treated with rapamycin or vehicle. There was no difference in leukemogenesis in primary recipients, but CD99 KO-AE9a AMLs exhibited a 72% (p=0.048) increase in protein synthesis compared with WT-AE9a AMLs (Figure 1A), confirming that CD99 negatively regulates protein synthesis in AML. We next performed secondary transplants to assess LSC function, as measured by survival of secondary recipients in the absence of rapamycin treatment (Figure 1B). We furthermore performed these transplants at limiting dilution to quantify LSCs (Figure 1C). CD99 KO-AE9a vehicle treated AMLs demonstrated improved survival and a lower LSC frequency compared with WT-AE9a vehicle treated AMLs, consistent with a self-renewal defect with loss of CD99. Rapamycin treatment completely rescued this defect, leading to decreased survival and increased LSC frequency in CD99 KO-AE9a AMLs compared with vehicle. Conversely, rapamycin treatment depleted LSCs in WT-AE9a AMLs, increasing survival and decreasing LSC frequency compared with vehicle. Thus, similar to HSCs, LSCs are adversely affected by both increases or decreases in protein synthesis.MLL-AF9-induced mouse AMLs initiated in HSCs as compared with granulocyte macrophage progenitors (GMPs) exhibit increased epigenetic imprinting of HSC features resulting in disease features reminiscent of high-risk AML (PMID: 23235717). To test if HSC-derived leukemias exhibit increased dependence on regulated protein synthesis, we transduced HSCs or GMPs from CD99 KO or WT mice to express MLL-AF9 and transplanted them into WT recipients, followed by secondary transplants to assess LSC function. Loss of CD99 led to increased survival indicative of decreased LSC function in HSC-derived but not GMP-derived leukemias (Figure 1D). This suggests that HSC-derived leukemias co-opt from HSCs a more pronounced dependence on tightly regulated protein synthesis. Accordingly, WT HSC-derived leukemias exhibited decreased protein synthesis as compared with their WT GMP-derived counterparts (Figure 1E), as well as increased sensitivity to rapamycin (Figure 1F).To directly study protein synthesis in human LSCs, we transduced primary AML specimens (n=4) to express a destabilized form of GFP (dGFP) from a constitutive promoter followed by xenotransplantation (Figure 1G), allowing us to measure dGFP by flow cytometry as a surrogate for protein synthesis rates in vivo. We validated this assay by measuring protein synthesis using orthogonal O-propargyl-puromycin incorporation assays (Figure 1H). Human AML cells with low levels of dGFP demonstrated increased engraftment in secondary transplants (Figure 1I), demonstrating that human LSCs exhibit low protein synthesis rates.In conclusion, our data demonstrate that LSCs co-opt from HSCs a dependence on tightly regulated protein synthesis. This is the first description of a cellular feature co-opted from HSCs that also represents a therapeutic vulnerability. Furthermore, the types of AML that exhibit the most robust re-activation of HSC programs and increased dependence on regulated protein synthesis are also likely to represent high-risk AMLs most resistant to standard therapies. Our data suggest that such therapy resistant AMLs may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Relevance of Alternatively Spliced Isoform AML1-ETO9a in Adult Patients with t(8;21)(q22;q22.1) Acute Myeloid Leukemia (AML): A Study of the German-Austrian AML Study Group (AMLSG)

Background: AML with t(8;21)(q22;q22.1) defines a distinct disease entity within the WHO category ‘AML with recurrent genetic abnormalities’ and results in the formation of the AML1-ETO (AE) fusion transcript. Recent data showed that alternative splicing generates a truncated variant of AE, namely AML1-ETO9a (AE9a), containing an additional exon 9a. Co-expression of AE and AE9a was shown to induce a more aggressive leukemic phenotype with a rapid onset of AML in a retrovirally transduced mouse model (Yan et al., Nat Med 2006). However, the clinical relevance of the alternatively spliced AE9a isoform in adult patients (pts) with t(8;21) AML is not well determined yet.Aim: We sought to assess the incidence and prognostic significance of AE9a co-expression in the context of clinical and genetic factors in a large clinically well-annotated cohort of pts with t(8;21) AML.Methods: 129 pts were included based on the availability of a diagnostic bone marrow (BM) or peripheral blood (PB) sample; all pts were either enrolled on an AMLSG clinical trial protocol (n=93) or received standard intensive chemotherapy regimens. AE9a mRNA transcript levels (TL) were determined by quantitative reverse transcription PCR (qRT-PCR) using TaqMan technology. Co-expression of AE9a as a fraction of the full-length AE transcript was reported as AE9a / AE ratio (%). ABL gene expression was used as housekeeping control. Additional gene mutation data were available for KIT, FLT3 -ITD/TKD, NRA S, and ASXL2 .Results: The median age of the pts was 49 years (yrs; range, 18-75 yrs); the alternatively spliced isoform AE9a was identified in all pts; the AE9a / AE ratio (range 3%-77%, median 32%) did not significantly differ between BM (n=116, range 8%-77%, median 31%) and PB (n=13, range 3%-66%, median 52%). Median AE9a / AE ratio was not correlated with clinical features (sex, age, WBC, platelets, BM blasts) or gene mutations affecting KIT (p=0.10), FLT3 -ITD/TKD (p=0.77) or ASXL2 (p=1.00); pts with an additional NRAS mutation showed higher AE9a / AE ratios as compared to wild-type pts (median, 44% vs 31%) without reaching statistical significance (p=0.06). Using Cox regression analysis, AE9a / AE ratios were not associated with the clinical endpoints overall survival (OS), event-free survival (EFS) and cumulative incidence of relapse (CIR). The same remained true when performing univariate analyses comparing AE9a / AE ratios dichotomized at the median (AE9a / AE>median vs AE9a / AE≤median): AE9a / AE had no impact on 4-yr OS (71% vs 79%; p=0.37), 4-yr EFS (59% vs 67%; p=0.83), and 4-yr CIR (32% vs 33%; p=0.35). Correlation of AE9a / AE ratios according to quartiles of distribution did also not affect OS (p=0.14), EFS (p=0.67), and CIR (p=0.70). Finally, we evaluated a possible correlation between AE9a / AE ratio and NRAS or KIT mutations which frequently co-occur in this AML subtype. In a subgroup analysis according to KIT or NRAS mutation status, no significant differences with regard to the clinical endpoints OS, EFS and CIR were found between AE9a / AE>median and AE9a / AE≤median.Conclusion: Alternative splicing of the AML1-ETO fusion transcript is a common feature in pts with t(8;21) AML. This finding is also supported by a recent collaborative transcriptome study where novel high-throughput sequencing (HTS) technologies were applied (Faber et al., Nat Genet 2016). Using different explorative approaches, AE9a has no impact on clinical outcome which precludes its potential as a novel independent prognostic marker in this AML subtype.MA was supported by the Else Kröner-Forschungskolleg. DisclosuresKindler:Novartis: Membership on an entity's Board of Directors or advisory committees. Lübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid. Fiedler:Amgen, Pfizer: Research Funding; Amgen, ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Patents & Royalties; Amgen, Gilead, GSO, Teva, Jazz Pharmaceuticals: Other: Support for meeting attendance. Götze:Celgene: Honoraria; Novartis: Honoraria; BMS: Honoraria; Abbvie: Honoraria. Griesshammer:Gilead: Consultancy, Honoraria, Speakers Bureau; Baxalta: Consultancy, Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; AOP Orphan: Consultancy, Honoraria, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Greil:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding; Novartis, Celgene: Research Funding; Takeda: Honoraria, Research Funding; BMS, Amgen: Honoraria. Döhner:Amgen: Honoraria; Celator: Honoraria; Pfizer: Research Funding; Seattle Genetics: Honoraria; Bristol Myers Squibb: Research Funding; Abbvie: Honoraria; Sunesis: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Honoraria; Boehringer Ingelheim: Research Funding; Astex Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Arog Pharmaceuticals: Honoraria, Research Funding. Döhner:Novartis: Honoraria, Research Funding.

Characterization of Cooperating Mutations in Murine Acute Leukemias: Towards Establishing Leukemia Models with Genetic Lesions in Defined Cellular Pathways

Acute leukemia (AL) is caused by somatic genetic alterations including chromosomal aberrations and point mutations. AL is genetically extremely heterogeneous with each case having about 4 to 8 cooperating driver mutations. Even with modern gene-editing techniques, like CRISPR-Cas9, it is difficult to generate animal models that have several driver mutations. We established retroviral transduction murine bone marrow transplantation leukemia models (MBMTLM) using two primary driver oncogenes and identified cooperating mutations using whole exome sequencing (WES). One model used the AML1-ETO9a (AE9a) fusion, an alternatively spliced isoform of AML1-ETO, and the second model used the CALM-AF10 minimal fusion protein (CA-MF), which contains the regions of CALM and AF10 required for leukemogenesis. AML1-ETO is associated with acute myeloid leukaemia (AML), whereas CALM-AF10 is seen in AML, T cell acute lymphoblastic leukemia and malignant lymphoma. Previous studies in animal models have shown that these fusion genes are not sufficient to cause leukemia on their own but require the cooperation of additional mutations. All of the AE9a (n= 13) and CA-MF (n=21) transplanted mice developed AL with a median latency of 115 days and 91 days, respectively. Immunophenotypic analysis of the AE9a leukemias showed a myeloid phenotype. Interestingly, immunophenotypic analysis of the CA-MF leukemias showed two distinct phenotypes: myeloid and biphenotypic leukemia. Both leukemias were transplantable into secondary and tertiary recipient mice, which showed the same leukemia phenotypes as their respective primary leukemias but had a much shorter latency.We hypothesised that the difference in phenotypes and latencies were due to different cooperating mutations which had arisen spontaneously. To identify these cooperating mutations, we performed WES on leukemia cells and the corresponding germline DNA of the donor mice. So far, we have analysed 32 leukemia exomes with a 97x mean coverage (15 AE9a and 17 CA-MF). On average, we found 9.1 somatic potential driver mutations per exome with significantly more mutations in AE9a leukemias than in CA-MF, 14.4 vs 3.8 per exome, respectively. Any somatic mutation with a high (nonsense) or moderate (missense) predicted impact was considered a potential driver mutation. In both CA-MF and AE9a leukemias, we found mutations in genes known to be mutated in human leukemia. One of the CA-MF leukemias had a mutation in the activation loop of the tyrosine kinase domain of Flt3 , while another carried a mutation in the RING finger domain of Cbl , which encodes an E3 ubiquitin ligase and is a negative regulator of FLT3 signaling. Mutations in the protein tyrosine phosphatase Ptpn11 and the polycomb group tumor suppressor gene L3mbtl1 (histone methyl-lysine binding protein) were found in the AE9a leukemias. The mutations found in Cbl , Flt3 and Ptpn11 correspond to known mutations in human leukemia. Even though we have only analyzed 24 leukemias so far, we found one recurringly mutated gene. Two AE9a leukemias and one CA-MF leukemia had mutations in the Ppig gene. Ppig encodes a peptidyl-prolyl cis trans isomerase. The mutated genes belong to the following functional groups: protein tyrosine phosphatases (Ptpn11, Ptprt , Ptprg) , tyrosine kinases (Flt3 , Sphk2 ), ubiquitin ligases (Cbl and Ubc9 ), transcriptional repressors or epigenetic modifiers (Dpy30 , Spen , L3mbtl1 ), nuclear exporters (Xpo1 , Ranbp3 ), guanine exchange factors (Dennd1b and Mcf2 ), DNA replication and DNA damage repair genes (Rfc3 , Rad54l2 ) and cohesin complex (Pds5a ). Members of these gene categories are known to be frequently mutated in human leukemia.Our results strongly suggest that CALM-AF10 and AML1-ETO require additional cooperating events to cause leukemia. MBMTLMs are a unique resource for studying clonal evolution and cooperating mutations. These genomically well characterised mouse leukemias provide more realistic models for human disease and can also lead to the discovery of potentially important novel pathways in leukemogenesis (eg: peptidylprolyl cis-trans isomerases). Our MBMTLMs will be a unique resource for the in depth study of leukemias with multiple driver mutations in defined cellular pathways (eg: growth factor signaling, nuclear export, cohesins, polycomb complex) and should facilitate development of treatment strategies targeting these pathways and combinations of pathways. DisclosuresNo relevant conflicts of interest to declare.

CD99 Regulates Normal and Malignant Stem Cell Function By Restricting Protein Translation

We recently reported that CD99 is highly expressed on disease initiating stem cells in myeloid malignancies. In acute myeloid leukemia (AML), high expression of CD99 identified human leukemia stem cells (LSCs) with enhanced leukemia-initiating capacity and gene signatures associated with hematopoietic stem cell (HSC) self-renewal. LSCs with high CD99 expression were also marked by significant depletion of ribosomal protein transcripts, and we have subsequently confirmed that they exhibit reduced levels of protein translation as measured by polysome profiling.To elucidate the mechanisms by which CD99 promotes self-renewal in hematopoietic cells, we characterized CD99-deficient mice generated by gene trapping (B6- CD99Gt(pU-21T)44lmeg , or CD99 Gt/Gt). At steady-state, homozygous CD99 Gt/Gt mice exhibit normal CBCs, bone marrow (BM) cellularity, and hematopoietic stem and progenitor frequencies. To test the function of CD99 Gt/Gt HSCs, we FACS-purified HSCs (Lin-Sca-1+c-Kit+CD34-CD150+) from CD99 Gt/Gt and wild-type (WT) mice and transplanted them into lethally irradiated WT recipients. At early time points, CD99 Gt/Gt HSCs and WT HSCs engrafted equally well. However, over the course of 24 weeks, there emerged in mice transplanted with CD99 Gt/Gt HSCs a strong trend towards decreased peripheral blood (PB) donor chimerism, as well as a progressive and highly significant decline in granulocyte (Mac-1+Gr-1+) donor chimerism, suggesting that CD99 Gt/Gt HSCs may gradually exhaust after transplantation (Fig.1A). BM evaluation at 24 weeks revealed no differences in HSC donor chimerism, but there was a significant decrease in myeloid progenitor (Lin-Sca-1-c-Kit+) donor chimerism and BM cellularity in mice transplanted with CD99 Gt/Gt HSCs (Fig.1B), consistent with decreased HSC functional output. In non-competitive secondary transplants using unfractionated BM, CD99 Gt/Gt HSCs exhibited a marked decrease in engraftment, consistent with impaired self-renewal.To assess the impact of CD99-deficiency on transcriptional output, we performed RNA-seq on HSCs from CD99 Gt/Gt and WT mice, identifying 327 differentially expressed genes (p<0.01). Gene set enrichment analysis identified an increase in transcripts associated with the proteasome, mTOR signaling, and c-MYC targets, as well as depletion of ribosomal protein transcripts (Fig.1C). This gene expression pattern suggests that CD99 Gt/Gt HSCs exhibit proteotoxic stress, which has been shown to promote transcriptional repression of ribosomal proteins.Given that tightly regulated translation has been shown to be essential for HSC function, as well as our findings in HSCs and LSCs that suggest that CD99 negatively regulates translation, we hypothesized that CD99 promotes self-renewal in HSCs by limiting protein translation. To test this, we transplanted HSCs from CD99 Gt/Gt and WT mice as before and treated recipient mice with the mTOR inhibitor rapamycin (4 mg/kg daily). Rapamycin completely rescued the engraftment defect of CD99 Gt/Gt HSCs at eight weeks (Fig.1D), strongly suggesting that their functional defects are mediated by increased translation.Finally, we measured CD99 expression on leukemia cells from mouse AML models including MLL-AF9 , AML-ETO9a , and NUP98-HOXD13, findingCD99 to be overexpressed compared with normal myeloid progenitors (Lin-Sca-1-c-Kit+), similar to our prior observations in human AML. Because AML1-ETO is correlated with high CD99 mRNA expression in human AML (ECOG 1900 cohort), we tested whether CD99 is required for AML1-ETO induced leukemogenesis. c-Kit+ cells from CD99 Gt/Gt and WT mice were transduced with AML1-ETO 9a (AE9a) and transplanted into lethally irradiated recipients. While there was no significant difference in survival between mice transplanted with CD99 Gt/Gt-AE9a vs. WT-AE9a cells (81 d vs. 108 d, p=0.32), CD99 Gt/Gt-AE9a grafts exhibited less splenomegaly and a dramatic reduction in c-Kit+ cells (43-fold, p=0.0064), suggestive of increased differentiation of leukemia cells and depletion of LSCs (Fig.1E). Consistent with this, RNA-seq revealed in CD99 Gt/Gt-AE9a cells depletion of HSC/LSC-associated gene signatures, as well as enrichment for ribosomal protein transcripts (Fig.1F).Together, these findings reveal an essential role for CD99 in HSC and LSC function, suggesting that it promotes self-renewal in part by acting as a key regulator of protein translation. [Display omitted] DisclosuresLevine:Roche: Research Funding; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding. Park:Forty Seven, Inc.: Consultancy.

Loss of Phospholipase C Gamma 1 (PLCG1) Impairs AML1-ETO Induced Leukemia Stem Cell Self-Renewal

Chromosomal translocations found in acute myeloid leukemia (AML) can generate oncogenic fusions with aberrant epigenetic and transcriptional functions. However, direct therapeutic targeting of leukemia fusion proteins has not been accomplished so far. Although high remission rates can be induced in patients diagnosed with AML1-ETO/t(8;21)-positive AML only half of them achieve long-term disease-free survival (Papaemmanuiel et al., NEJM, 2016). In the other half of these patients, the disease maintaining leukemia stem cell (LSC) clone is not eliminated by chemotherapy. A functional characteristic of LSCs is unlimited self-renewal capacity and several signaling pathways have been identified that maintain stem cell self-renewal. Targeting the oncogene induced self-renewal capacity of LSCs has great potential to eliminate the malignant clone and prevent relapse. To identify oncogenic cellular functions with relevance for LSC self-renewal, we performed global proteome profiling in murine AML1-ETO9a (AE) compared to MLL-AF9 (MA9) driven LSCs. Gene set enrichment analyses revealed a significant enrichment of calcium-dependent cellular functions and Phospholipase C (PLC)-signaling in AE LSCs. These data could be confirmed in sorted CD34+ blasts from AE-positive AML when compared to non-AE-AML. All PLC family members are regulators of Ca2+ homeostasis. However, when analyzing published AML gene expression datasets we found exclusively PLCG1 to be highly expressed in t(8;21) AML. Conditional activation of AE in embryonic stem cells resulted in induction of PLCG1 expression and PLCG1 was identified as a direct target of the AE fusion by ChIP-sequencing in AE-positive Kasumi-1 cells.Here, PLCG1 depletion resulted in reduced Ca2+ release, impaired proliferation and reduced colony formation in vitro. In a xenograft model, inactivation of PLCG1 resulted not only in delayed disease development (median survival shNT vs. shPLCG1: 135 days vs. not reached, p=0.02) but also in reduction of disease penetrance by 87%. Consistent with these results, transcriptome analysis revealed strong induction of gene sets related to myeloid differentiation and down-regulation of gene sets linked to proliferation, stemness and c-Myc targets. To confirm the functional role of PLCG1-signaling in AE-driven LSCs, we generated a new conditional knockout mouse model for Plcg1 and induced leukemia using the oncogenes AE and KRAS-G12D (AE/K). Genetic inactivation of Plcg1in vivo after engraftment of leukemic cells resulted in significant reduction of LSC numbers (p=0.04) and a reduction of disease penetrance by 67% in primary recipients. Isolated LSCs revealed induction of differentiation, loss of cell cycle activity and failed to re-establish disease in secondary recipients (Plcg1+/+ vs. Plcg1-/-: median survival 12 days vs. not reached; p=0.0001). In contrast, genetic deletion of Plcg1 appeared to be dispensable for normal murine HSC function during primary and secondary transplantation. Primary human t(8;21) AML cells (derived from 4 different donors) showed impaired colony forming capacity following PLCG1 inactivation in vitro irrespective of co-occurring mutations while colony formation of human CD34+ BM cells was not affected to a major extent. As Ca2+ signaling appeared deregulated in t(8;21) AML, we aimed to investigate the effects of pharmacologic Ca2+ inhibition as a tractable target downstream of PLCG1. To assess specifically for LSC function, we treated primary recipient mice with established AE/K-driven leukemia with the clinically approved calcineurin inhibitor ciclosporin (CsA), a compound that blocks intracellular Ca2+ release. CsA-treated animals showed reduction in total leukemic burden (spleen weight diluent vs. CsA, p=0.01) and LSC numbers (p=0.02). This resulted in increased survival of secondary recipient hosts (diluent vs. CsA: median 15 vs. 29 days, p=0.0002). These effects could not be observed for other oncogenes (e.g. MA9), confirming its specificity for AE-induced disease. Consistently, CsA treated primary human t(8;21)-positive AML blasts failed to form colonies in methylcellulose. In summary, our findings identified PLCG1-dependent Ca2+ signaling as a critical pathway for t(8;21) LSC maintenance and self-renewal. Most importantly, as PLCG1 is dispensable for maintenance of normal HSPCs, PLCG1 could serve as a novel therapeutic target in t(8;21) AML. Disclosures Döhner: Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bullinger:Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Abbvie: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Bayer: Other: Financing of scientific research; Pfizer: Honoraria.

Selective Dependency of MLL-Rearranged Leukemia on Immunoproteasome Function

Several cellular pathways control the fine balance between self-renewal and differentiation to maintain leukemia-initiating cell (LIC) function. To identify cellular dependencies with relevance for oncogenic fusion proteins, we performed global proteome profiling. Acute myeloid leukemia (AML) was induced by retroviral expression of either MLL-AF9 (MA9) or AML1-ETO9a (AE) in murine hematopoietic stem and progenitor cells (HSPCs) (Lineage-Sca1+Kit+, LSK) which were subsequently transplanted into irradiated syngeneic recipients. After onset of leukemia, LIC-enriched (GFP+ Kithigh) cells isolated from 4 different primary recipients (per oncogene) were analyzed by in-depth quantitative proteomic analysis using high-resolution mass spectrometry (MS). More than 3,000 proteins were quantified with 868 proteins being differentially expressed between MA9 and AE LIC-enriched populations. In MLL-rearranged (MLLr) cells, gene set enrichment analysis (GSEA) revealed significant enrichment of cellular functions related to protein degradation and proteasome function. As this enrichment is present in MLLr-leukemia but not AE-driven LICs, may indicate an oncogene specific vulnerability. Expression of proteasome subunits is highly heterogeneous between different cell types and therefore may also be influenced by the underlying differentiation stage or oncogenic fusion. In published AML gene-expression datasets, immunoproteasome (IP) subunits PSMB8/LMP7 (p=0.0003***), PSMB9/LMP2 (p=0.0007***) and PSMB10/MECL1 (p&amp;lt;0.0001****) showed significantly higher expression in MLLr compared to non-MLLr-AML. IP is a proteasomal variant constitutively expressed in cells of hematopoietic origin, induced under stimulation with pro-inflammatory cytokines and relevant for mediating stress-responses during inflammation and infection. To assess for functional dependency of MLLr cells on IP subunits we performed an in vitro CRISPR/Cas9 dropout screen in MLLr MOLM-13 cells. Genetic inactivation of PSMB8/LMP7 resulted in outcompetition with 3/5 sgRNAs, while there was less dependency detectable for the other subunits. Specificity of this finding was confirmed in 5 different cell lines (4 MLLr; 1 non-MLLr) by RNAi using 2 shRNAs against PSMB8/LMP7 versus non-targeting control. To confirm these findings in primary cells, we used a previously published conventional LMP7 knockout mouse model (Fehling et al., Science, 1994). LSK cells sorted from the bone marrow (BM) of LMP7 knockout and wildtype mice were retrovirally transformed with either MA9, MLL-ENL (ME) or NUP98-HOXA9 (as non-MLLr control) to assess for disease development by serial plating in methylcellulose. Only in MA9 or ME transformed cells LMP7-deficiency limited re-plating capacity to 2-4 rounds. When we injected 2,5x 104 MA9-infected LSK cells into sublethally irradiated recipient mice, recipients of MA9-LMP7-/- cells (n=12) and MA9-LMP7+/+ (n=12) showed development of AML. However, recipients of MA9-LMP7-/- cells had a significant delay in AML development (median survival 63.0 days for LMP7+/+ versus 92.5 days for LMP7 -/- animals, p=0.0387*). Besides the significant delay in AML development, disease penetrance was reduced by 50%, indicating that deficiency for LMP7 impairs development of MA9 driven AML. In contrast, immunophenotypic abundance of HSPCs in LMP7-/- versus LMP7+/+ animals revealed comparable numbers in all relevant subpopulations. Competitive transplantation of LMP7-/- BM into recipient hosts showed no competitive disadvantage or lack of self-renewal capacity compared to LMP7+/+ controls. Pharmacologic inhibition of IP function using the specific LMP7-inhibitor PR-957 (ONX-0914) resulted in significant delay of disease development in secondary recipient hosts. To assess its effect on LIC frequency we performed limiting dilution assays of MA9 leukemic cells in sublethally irradiated recipient mice. PR-957 treatment reduced LIC frequency compared to DMSO control (1/57410 vs. 1/4450). Pharmacologic inhibition of PSMB8/LMP7 in human MLLr leukemia cell lines induced cellular differentiation. Likewise, cell cycle and metabolism appeared affected, functions which could be confirmed by global transcriptome analysis. Taken together, our studies uncover a selective dependency of MLLr-leukemia on IP function and identify PSMB8/LMP7 as a tractable target. Disclosures Heidel: Celgene: Consultancy; Novartis: Consultancy, Research Funding; CTI: Consultancy.

Downstream Pathway of AML1-ETO Targeted ARHGEF12 Might be Targetable for Rock Inhibitor to Improve Therapy Outcome

As a transcription factor AML1-ETO modulate gene expression of its targets. Scanning our ChIP-seq data we found AML1-ETO bind to an AML1 binding motif in the promotor region of ARHGEF12. Exploring acute myeloid leukemia (AML) microarray databases we confirmed that the expression of ARHGEF12 was up-regulated consistently in AML1-ETO-positive AML patient samples. To confirmed this finding, we quantitated expression of ARHGEF12 in pediatric AML samples from Shanghai children's medical center. Not out of expectation, all AML1-ETO-positive cases have higher ARHGEF12. Moreover, the ARHGEF12 expression was an independent poor prognostic factor for overall survival comparing between patients with higher and lower expressed ARHGEF12 (P=0.0258) (Figure A).To understand the role of ARHGEF12 in leukemogenic function of AML1-ETO fusion gene, we knocked down ARHGEF12 expression in kasumi-1 cells by shRNA. Growth of kasumi-1 cells in vitro was significantly inhibited with cell cycle exit and more apoptosis. Further, we transformed fetal liver cells of Arhgef12 conditional knockout mice to leukemia model by retrovirus conducted AML1-ETO9a(AE9a) expression. Induced Arhgef12 deletion delayed full blown of leukemia in vivo. The overall survival prolonged 15.5 days (median 83.5 vs. 68 days, P=0.0072). (Figure B) ARHGEF12 is a well-known guanine nucleotide exchange factor for RhoA GTPase. Arhgef12 deletion decreased activated GTP-RhoA and MYPT1 phosphorylation, which is one of ROCK kinase substrates. Spleen cells from leukemic mice were cultured in vitro, the ROCK inhibitor Y-27632 significantly decreased the cell proliferation, which mimic the tamoxifen-induced Arhgef12 knockout phenotype. Y-27632 combined with tamoxifen treated cells almost eradicate alive leukemia cells on the ninth day in vitro (Figure C) and delay leukemogenesis in vivo (Figure D).These findings suggest that ARHGEF12 as a transcriptional target of the AML1-ETO fusion protein and plays an essential role in leukemogenesis. The ARHGEF12-RHOA-ROCK pathway may serve as a new therapeutic target for AML1-ETO+ AML [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Caspase-3 controls AML1-ETO–driven leukemogenesis via autophagy modulation in a ULK1-dependent manner

AbstractAML1-ETO (AE), a fusion oncoprotein generated by t(8;21), can trigger acute myeloid leukemia (AML) in collaboration with mutations including c-Kit, ASXL1/2, FLT3, N-RAS, and K-RAS. Caspase-3, a key executor among its family, plays multiple roles in cellular processes, including hematopoietic development and leukemia progression. Caspase-3 was revealed to directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3–compromised background and thereby accelerate leukemogenesis. Therefore, we developed a Caspase-3 knockout genetic mouse model of AML and found that loss of Caspase-3 actually delayed AML1-ETO9a (AE9a)-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation and/or progression of AML. We report here that loss of Caspase-3 triggers a conserved, adaptive mechanism, namely autophagy (or macroautophagy), which acts to limit AE9a-driven leukemia. Furthermore, we identify ULK1 as a novel substrate of Caspase-3 and show that upregulation of ULK1 drives autophagy initiation in leukemia cells and that inhibition of ULK1 can rescue the phenotype induced by Caspase-3 deletion in vitro and in vivo. Collectively, these data highlight Caspase-3 as an important regulator of autophagy in AML and demonstrate that the balance and selectivity between its substrates can dictate the pace of disease.

A Novel CD34/ETO2/Ifngr gene Regulatory Axis Is Implicated in Poor-Prognosis Cases of t(8;21) AML

Abstract Background:Roughly 15% of AML cases are derived from the t(8;21) translocation, a molecular event that results in the production of the AML1-ETO (AE) fusion transcription factor. This aberrant protein induces a broad dysregulation of the transcriptome and causes expansion of leukemia stem cells (LSCs), which predisposes clones to an increased risk of second-hit mutations. In isolation, without the presence of type I mutations, the t(8;21) mark is considered to be one of favorable prognosis. However, a number of LSC characteristics have been shown to influence patient survival, including expression levels of the cell surface glycoprotein, CD34, and of the truncated isoform of AE, AML1-ETO9a (AE9a). Despite these findings, a unified model of how these factors interact to further the progression of t(8;21) AML has not been determined. Here, we use a combination of in vitro assays and bioinformatic analyses of patient datasets with the hopes of identifying an underlying connection between independent, prognostic factors. Results: We first performed gene knockdown studies with shRNA constructs (against AE) in a pKLO.1 backbone, which was transduced into the AML-model cell line Kasumi-1. Subsequent RT-PCR showed a large decrease in CD34 expression and, serving as a control, an increase in RASSF2, a cytoplasmic signaling protein normally repressed by AE. This led us to perform gene overexpression of cDNA encoding AE or AE9a into CD34-enriched cord blood HSCs. Interestingly, we were able to show that both AE and AE9a increased the expression of CD34, and AE9a was able to do so to a greater extent. This prompted us to analyze previous ChIP-seq data from Kasumi-1 cells, and we discovered high levels of AE binding to an upstream enhancer of CD34. While this site was free of any E-box binding motifs, we nonetheless observed binding peaks of the E-protein HEB, and further gene knock-down studies showed that HEB recruitment to this site depended on AE and, in turn, CD34 expression depended on HEB recruitment. Next, we used differential gene expression analyses to determine the CD34-coexpressed genes from a publically available patient microarray dataset (GSE14468, n=516). CD34-high patient samples showed an accompanying enrichment of JUP, KIT, CD133, HEB, E2A and ETO2, and a decrease in immune signaling molecules such as IFNGR1 and IFNGR2. ETO2 is the only ETO family member significantly expressed in HSCs and is an important corepressor of E-proteins HEB and E2A; it is thought to help repress the pro-differentiation and anti-proliferation effects of E-proteins. ETO2, being an ETO family member, is also able to oligomerize with other ETO family members, such as ETO, and thus may play a role in modifying the functions of the AE-transcriptional complex. Intriguingly, we have evidence that AE and AE9a repress ETO2, although AE9a does so to a lessened extent. We then performed Kaplan-Meier analyses on subgroups of non-CBF AML and t(8;21) AML cases. Unsurprisingly, we found that both groups experienced lower event-free survival when CD34 and ETO2 were high (p=.028 and p=.040), and the t(8;21) group when IFNGR was low (p=.014). Additionally, although CD34 expression was higher in t(8;21) patients compared to non-CBF AML (p=.0027), ETO2 expression was lower (p=.025). If both subgroups were further divided into CD34-high and CD34-low groups, ETO2 expression was, in both cases, higher in the CD34-high patients and lower in the CD34-low patients, suggesting a possible regulatory link between these two genes. Finally, we performed ETO2 gene knockdown experiments in CD34+ HSCs and observed an increase in IFNGR1 and IFNGR2 expression. Conclusions: Our findings suggest a clinically relevant association between CD34, ETO2, and IFNGR in both t(8;21) and non-CBF AML. We believe this regulatory axis is especially relevant in relapse likelihood and may be hijacked by LSCs to preserve their "stemness" and resistance to therapy. Our findings may suggest that AE simultaneously activates CD34 and represses ETO2, providing explanation for why full-length AE is slow to induce leukemia in mouse models and a relatively good prognostic mark in humans. Additionally, our findings suggest a loss of ETO2repression as a potential mechanism by which the AE9a isoform, and perhaps other second-hit mutations, converts LSCs to a more dangerous phenotype. Supported by NIH grants R01HL093195-01A1 to JZ and T32 training grant T32GM008306-26A1 Disclosures No relevant conflicts of interest to declare.

Caspase-3 Can Promote Acute Myeloid Leukemia Development By Regulation of Autophagy

BackgroundAML1-ETO (AE), a oncogenic protein generated by the t(8;21) translocation, causes acute myeloid leukemia (AML) in collaboration with other secondary events. The leukemogenicity of AE has been evaluated in multiple mouse models, such as expression of AE in Cdkn1a-null Hematopoietic stem cell (HSCs) and expression of AML1- ETO9a (AE9a), an alternatively spliced variant of AE, in WT HSCs. Both lead to the development of fully penetrant AML.Caspase-3 plays multiple roles in hematopoietic development and leukemia progression and treatment by affecting proliferation, self-renewal and differentiation. It has been shown that uncleaved caspase-3 levels are higher in the peripheral blood cells of AML patients compared to normal individuals, which suggests that the caspase pathway is dysregulated in AML. We and others have shown that Caspase-3 directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3 compromised background and accelerate leukemogenesis.MethodsWe developed a Caspase-3 knockout genetic mouse model of AML based on fetal liver cell transplantation. In brief, fetal liver cells from WT or Caspase3-/- mice were transduced to express AE9a in vitroand 100,000 AE9a+ transduced cells were transplanted into lethally irradiated recipient mice by tail-vein injection.ResultsWe found loss of Caspase-3 impaired leukemia stem cell (LSC) self-renewal and delayed AE9a-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation or progression of AML.Moreover, we identified a new substrate of Caspase-3, ULK1, by in vitro cleavage assays and site-directed mutagenesis. ULK1 (serine/threonine UNC-51-like kinase) is the homology of Atg1 (the first autophagy related gene found in 1997) in mammalian cells, which is a direct target of mTOR and is responsible for initiation of the autophagic activity by forming a complex with mAtg13, FIP200 and Atg101. The induction of autophagy caused by upregulation of ULK1 in AE/AE9a-expressing Caspase-3-/- fetal liver cells acted to limit the leukemogenicity of AE9a in vivo. Inhibition of ULK1 by inhibitor or shRNAs could rescue the self-renewal capability induced by Caspase-3 deletion in serial replating assays.Unexpectedly, when we expressed AE/AE9a in fetal liver cells from WT and Caspase-3-/- mice, the protein levels were comparable suggesting the basal level of Caspase-3 didn't affect the expressing of AE/AE9a in fetal liver cells.ConclusionAutophagy may play a general role in the development and treatment of leukemia. In human AML, blasts display reduced expression of autophagy-related genes and decreased autophagic flux, indicating that low autophagy activity provides a general advantage for leukemia development. Beside this, a number of chemotherapy drugs have been reported to be able to induce leukemia cell death via activation of autophagy suggesting that autophagy plays critical roles in the leukemia treatment.Our study reveals that Caspase-3 regulates autophagy through its direct cleavage of ULK1 and this interaction dictates the pace of AE-driven leukemogenesis. Targeting this pathway may have therapeutic benefit for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

The PARP Inhibitor Olaparib Antagonizes Leukemic Growth Induced By TET1 Overexpression in AML1-ETO Positive Acute Myeloid Leukemia

DNA methylation patterns are highly deregulated in human acute myeloid leukemia (AML) cases and stratify AML patient samples into different subgroup. AML1-ETO is the most commonly occurring fusion gene in AML and these AML cases exhibit an aberrant and distinct methylation pattern. So far, the underlying mechanisms for this are only poorly understood. The TET1 dioxygenase has recently emerged as an important epigenetic modifier: by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) TET1 plays an important role in active demethylation, thereby regulating a variety of biological processes. It was linked to tumorigenesis based on the observation that its expression is frequently deregulated in solid cancer. However, the role of TET1 in AML1-ETO+ (AE+)human AML cases is yet unexplored. Using quantitative real time (qRT)- PCR we now show that AE+ AML is characterized by high and aberrant expression of TET1: the gene was significantly higher expressed in the majority of AE+ patients (n=7, p<0.01) compared to other AML subtypes such as inv(16) (n=11), PML-RARα+ (n=31), cytogenetically normal (CN)-AML patients (n=33) and CD34+ normal BM cells (n=4). This observation was consistent with published cDNA microarray data on large patient cohorts (Haferlach et al., JCO 2010, p<0.008 t-test, p<0.01 Anova) and recently published transcriptome data (TCGA) of AML patients. In contrast to TET1, TET2 and TET3 did not show significant higher expression in AE+ patients compared to other AML subtypes. In line with patient data, TET1 was highest expressed in the AE+ AML cell line KASUMI-1 and SKNO-1 compared to other AML cell lines (p<0.05 and n=3). Compared to normal CD34+ and myeloid (CD33+, CD15+ and CD14+) cells (n=3), TET1 was 10-fold and 16-fold higher expressed in AE+ patient samples (n= 7). Aberrant expression of TET1 in AE+ leukemic cells was associated with hypomethylation of its promoter and enrichment for H3K4me3 euchromatic marks at its promoter as determined by LC/MS and ChIP-qPCR respectively. Knockdown (KD) of TET1 mRNA using two short hairpin RNAs (shRNAs) in AE+ AML cell lines impaired their cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). shRNA mediated depletion of TET1 did not impact the cell growth and clonogenicity of the TET1 negative cell line RAJI, ruling out off target effects of the shRNAs (n=3). In mice, KD of Tet1 in leukemic bone marrow cells expressing the truncated leukemogenic AML1-ETO9a (AE9a) fusion, dramatically inhibited cell growth (>60% compared to scrambled, n=3, p<0.01), clonogenicity (>50-70% reduction in primary CFCs, p<0.01, n=3) and importantly delayed onset of leukemia in vivo (median survival 35 days for scr vs 80 days for shRNA mice, n=4/arm, p<0.03). Tet1-knock-out c-kit+ hematopoietic stem and progenitor cells (HSPCs) transduced with AE9a showed reduced primary colony formation and impaired serial replanting capacity in vitro compared to AE9a transduced Tet1-wild-type HSPCs (>50% and >70%, respectively; p<0.001, n=3). Global analysis of 5hmC and 5mC levels using hMeDIP/MeDIP-seq performed on TET1 depleted KASUMI-1 cells revealed lower global 5hmC levels and increase in 5mC as compared to cells transduced with scrambled control (n=2). 3324 promoter regions lost 5hmC and gained 5mC upon TET1 depletion (-5kTSS, Fold enrichment cut off <2-fold, q-value<1e5). Recent studies have shown that PARP activity induces TET1 expression by regulating its promoter epigenetically. We could show that aberrant TET1 expression could be antagonized by the PARP inhibitor olaparib in AE+ leukemic cell lines. Furthermore, olaparib treatment decreased 5hmC levels and reduced cell growth and clonogenicity of human AE+ cell lines and of the murine AE9a+ leukemic cell line in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1 expression contributes to the growth of AE+ AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML. DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML.We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2’-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed.Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis.*LH and NH contributed equally DisclosuresOgawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Rac1 GTPase Promotes Interaction of Hematopoietic Stem/Progenitor Cell with Niche and Participates in Leukemia Initiation and Maintenance in Mouse.

Interaction between hematopoietic stem/progenitor cells (HSPCs) with their niche is critical for HSPC function. The interaction also plays an important role in the multistep process of leukemogenesis. Rac1 GTPase has been found to be highly expressed and activated in leukemia patients. Here, by forced expression of constitutively active form of Rac1 (Rac1-V12) in HSPCs, we demonstrate that active Rac1 promotes interaction of HSPC with niche. We then established an active Rac1 associated acute myeloid leukemia (AML) model by expression of Rac1-V12 cooperated with AML1-ETO9a (AE9a) in mouse HSPCs. Compared with AE9a alone, Rac1-V12 cooperated with AE9a (AER) drives an AML with a short latency, demonstrating that activation of Rac1 GTPase in mice promotes AML development. The mechanism of this AML promotion is by a better homing and lodging of leukemia cells in niche, which further enhancing their colony formation, quiescence and preventing leukemia cells from apoptosis. Further study showed that an inhibitor targeting activated Rac1 can increase the efficacy of chemotherapeutic agents to leukemia cells. This study provides evidence that activation of Rac1 promotes leukemia development through enhancing leukemia cells' homing and retention in niche, and suggests that inhibition of Rac1 GTPase could be an effective way of eliminating AML cells. Stem Cells 2016;34:1730-1741.

JAK inhibitors suppress t(8;21) fusion protein-induced leukemia

Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein AML1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via down-regulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML.

Combined gene expression and DNA occupancy profiling identifies potential therapeutic targets of t(8;21) AML

AbstractChromosome translocation 8q22;21q22 [t(8;21)] is commonly associated with acute myeloid leukemia (AML), and the resulting AML1-ETO fusion proteins are involved in the pathogenesis of AML. To identify novel molecular and therapeutic targets, we performed combined gene expression microarray and promoter occupancy (ChIP-chip) profiling using Lin−/Sca1−/cKit+ cells, the major leukemia cell population, from an AML mouse model induced by AML1-ETO9a (AE9a). Approximately 30% of the identified common targets of microarray and ChIP-chip assays overlap with the human t(8;21)–gene expression molecular signature. CD45, a protein tyrosine phosphatase and a negative regulator of cytokine/growth factor receptor and JAK/STAT signaling, is among those targets. Its expression is substantially down-regulated in leukemia cells. Consequently, JAK/STAT signaling is enhanced. Re-expression of CD45 suppresses JAK/STAT activation, delays leukemia development, and promotes apoptosis of t(8;21)–positive cells. This study demonstrates the benefit of combining gene expression and promoter occupancy profiling assays to identify molecular and potential therapeutic targets in human cancers and describes a previously unappreciated signaling pathway involving t(8;21) fusion proteins, CD45, and JAK/STAT, which could be a potential novel target for treating t(8;21) AML.

The t(8;21) Fusion Protein AML1/ETO Promotes Susceptibility to Mutation In Acute Myeloid Leukemia.

AbstractAbstract 3368Translocation (8;21)(q22;q22) is the most common cytogenetic abnormality found in acute myeloid leukemia (AML), presenting in around 15% of all cases and is also reported in lymphoid leukemia. This subtype of AML has a favourable prognosis in contrast to other AML subtypes, however the mechanisms underlying this good response to treatment are not well understood. The translocation between AML1 (RUNX1) and the almost full-length ETO (RUNX1T1, MTG8) gene produces a 752 amino acid fusion protein and changes a transcriptional modulator essential for hematopoiesis into a constitutive repressor. When AML1/ETO (AE) is expressed in animal models, further mutations are required for the development of leukemia. AE has also been detected at a considerable frequency in the Guthrie cards of individuals who developed t(8;21) AML up to twelve years later, suggesting that AE+ hematopoietic precursor cells can remain latent for many years before transformation. AE+ cells have an activated p53 pathway, increased levels of p53 response genes and display an increased susceptibility to DNA damaging agents. AE also decreases the levels of DNA repair genes, including those involved in base-excision repair. This evidence predicts the existence of a pre-leukemic, AE-expressing hematopoietic precursor, which easily acquires additional mutations required for transformation, but that also responds well to treatment. There are multiple alternative forms of AE, the best described being AML1/ETO9a (AE9a), which codes for a truncated version of the protein lacking a significant proportion of ETO. AE9a has been found to be more leukemogenic than AE in mouse models by causing leukemia without the need for additional mutations.Our investigation aims to test the effect of cellular expression of AE or AE9a on mutation, cell death and transformation following exposure to leukemogens and to identify co-operating mutations involved in t(8;21) AML. TK6 lymphoblastoid cells are heterozygous for the TK gene and when exposed to mutagens, can lose their one functional copy and become TK-/-. These mutants can then be selected for and quantified. TK6 were lentivirally transduced with vectors containing EGFP and either AE or AE9a. AE was cloned into the pHR-SINcPPT-SIEW lentiviral transfer vector and a similar AE9a construct acquired. Virus was produced utilising the 293T cell line and TK6 were transduced by a spinfection technique. Expression was confirmed by flow cytometry, western blotting and qRT-PCR. Single clonal populations were generated and fusion gene expression levels of 28% (AE) and 40% (AE9a) relative to AE expression in Kasumi-1 (a t(8;21) AML patient-derived cell line) were achieved.We used these transduced cell line models to determine whether the presence of AE or AE9a affected cell viability and susceptibility to the acquisition of mutations. Spontaneous TK-/- mutants were first purged from the population by treatment with CHAT media. Two weeks post CHAT treatment, AE+ TK6 were shown to acquire a greater number of mutations than controls, both spontaneously and when exposed to doxorubicin (Figure 1). AE+ TK6 had a 2.2 fold higher background mutation frequency than controls, suggesting that AE promotes spontaneous acquisition of mutations. When treated with low-dose doxorubicin, AE+ TK6 had a 2–3 fold higher mutation frequency (3.5 × 10−5) in comparison to controls; wild-type TK6 and backbone transduced TK6 (1.4 × 10−5 and 1.1 × 10−5 respectively). High density SNP array analysis of AE-expressing TK6 has recently identified deletions at 10q25.1 and 3p24.3. The deletion at 3p24.3 potentially affects expression of SGOL1, coding for a protein involved in sister chromatid segregation and PCAF, an acetyl transferase which has been implicated in the regulation of p53. The effect of the deletion on these genes is currently being evaluated. [Display omitted] Our data show that TK6 expressing AE or AE9a have an enhanced sensitivity to the cytotoxic effects of the anthracycline doxorubicin. This contributes to the understanding of the relatively good prognosis of t(8;21) AML. We have shown that expression of AE in a cell system promotes the acquisition of more mutations than that of non-expressing cells. This provides further understanding as to how the pre-leukemic AE+ hematopoetic clone transforms and results in leukemia. Disclosures:No relevant conflicts of interest to declare.