ALBERT

Description: Congenital neutropenia (CN) is a rare inherited disorder of hematopoiesis with a 20% risk of evolving into acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Using next-generation sequencing in 31 CN patients who developed leukemia we found that 20 of the 31 patients (64.5%) had mutations in RUNX1 (runt-related transcription factor 1). Of these 20 patients, 19 had inherited mutations associated with CN. Intriguingly, the majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R (colony stimulating factor 3 receptor) mutations. Other leukemia-associated mutations (EP300, FLT3-ITD, CBL, and SUZ12) were less frequent. In eight patients, we detected two distinct heterozygous RUNX1 mutations. These mutations were localized to the splice-acceptor site of intron 4, affecting splicing of exons 3 and 4, which encode the Runt homology/DNA binding domain (RHD) of RUNX1, or solely in the RHD or were present in both RHD and trans-activation domain (TAD). In two patients, we were able to perform allele-specific analysis of RUNX1 mutations. Patient #10 had an Phe13TrpfsX14 deletion on one allele of RUNX1 and an Arg139ProfsX47 deletion on the other allele. In Patient #14, two RUNX1 mutations were on the same allele; one of the mutations (Met240Ile) was inherited from the mother and was localized two amino acids before the TAD, and the second acquired mutation (Arg139Gly) was in the RHD of RUNX1. Ten patients with RUNX1 mutations developed monosomy 7 and six patients developed trisomy 21 at diagnosis of leukemia. In contrast to their high frequency in CN evolving into AML, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric AML. RUNX1 mutations were mainly found in pediatric AML patients with an adverse prognosis. A sequential analysis at stages prior to overt leukemia in ten CN/AML patients showed that RUNX1 mutation is a late event in leukemogenic transformation. In 6 of 10 patients, a CSF3R mutation occurred prior to RUNX1 mutations (24-192 months prior to CN/AML for CSF3R mutations vs. 1-36 months prior to CN/AML for RUNX1 mutations). Interestingly, monosomy 7 or trisomy 21 appeared after acquisition of RUNX1 mutations and no additional chromosomal aberrations were detected by array-CGH. Single-cell analyses in two patients revealed that RUNX1 and CSF3R mutations were segregated in the same malignant clone. Moreover, functional studies demonstrated elevated G-CSF-induced proliferation with diminished myeloid differentiation of hematopoietic CD34+ cells after co-transduction with mutated RUNX1 and CSF3R, in comparison to cells transduced with mutated RUNX1 or mutated CSF3R only. The importance of RUNX1 mutations in leukemogenic transformation was substantially strengthened by the analysis of a unique family with two siblings suffering from CN that subsequently transformed to AML. In both children, cooperating RUNX1 and CSF3Rmutations were detected that were not present in healthy family members. Taken together, the high frequency and the time course of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia suggests a unique molecular pathway of leukemogenesis similar as has been reported in the Gilliland-Griffin two-hit hypothesis for AML development. The concomitant detection of RUNX1 and CSF3Rmutations represents a useful biomarker for identifying CN patients with a high risk of progressing to leukemia or MDS. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

Description: S.E. and J.K.K. as well as J.H.K. and M.M.H.E. contributed equally to this study. MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and in the development of leukemia. In addition, modulation of miRNA expression can be exploited therapeutically. To identify tumor suppressive miRNAs in pediatric acute myeloid leukemia (AML), we performed a large-scale miRNA expression profiling in 90 cytogenetically characterized, de novo AML cases using a RT-qPCR platform. In total, 253 miRNAs were significantly differentially expressed between patients with MLL rearrangements, t(8;21), inv(16), t(7;12), and t(15;17). Hierarchical clustering of patient samples using these sets of miRNA values showed that t(15;17) samples clearly cluster away from the other pediatric AML samples while t(7;12) patients cluster closely to core binding factor AMLs, (t(8;21) and inv(16). These three groups largely cluster away from the majority of MLL rearranged samples. Eight miRNAs specifically downregulated in MLL rearranged, t(8;21) and inv(16) AMLs were functionally evaluated in vitro using three cell lines representing those cytogenetic groups: THP-1 (MLL rearranged), KASUMI-1 (t[8;21]) and ME-1 (inv[16]). Two of two miRNAs tested in KASUMI-1 cells (miR-9 and miR-582), two of three miRNAs tested in ME-1 (miR-192/194 bicistron and miR-660) and one of three miRNAs tested in THP-1 (miR-181a/b bicistron) reduced cell growth and colony-forming capacity upon ectopic expression. In KASUMI-1 cells, one miRNA was identified, miR-9, that not only reduced cell growth and colony forming capacity but also strongly induced monocytic differentiation in concert with calcitriol without affecting apoptosis. During normal hematopoiesis miR-9 is only expressed in macrophages.The effects on cell growth, colony-forming capacity and differentiation were confirmed in a second AML cell line with t(8;21), SKNO-1. The differentiation induction was restricted to t(8;21) leukemic cell lines, while its growth inhibitory function was also evident in normal CD34+ hematopoietic stem and progenitor cells. Most strikingly, miR-9 exerted a tumor suppressive function in primary leukemic blasts from patients with t(8;21) (n=2), but not in patients with MLL rearrangements (n=3). Using global gene expression studies upon ectopic miR-9 expression, we identified and validated LIN28B and HMGA2 as high fidelity target genes of miR-9 by RT-qPCR, western blotting and luciferase reporter assays. LIN28B is known to suppress let-7 processing. Indeed, miR-9 overexpresion increased the levels of mature let-7 family members, also leading to HMGA2 downregulation. ShRNA-mediated downregulation of LIN28B or HMGA2 partially recapitulated the effects of miR-9 on proliferation and differentiation of t(8;21) cell lines. Thus, miR-9 is a tumor suppressor-miR in t(8;21) de novo pediatric AML, that acts in a stringent cell context dependent manner in concert with let-7 family members by repressing the oncogenic LIN28B/HMGA2 axis. This work was supported by grants to J.H.K. from the German National Academic Foundation (KL-2374/2-1) and to J.K.K., L.V., A.D.vO, C.M.Z. and M.M.H.-E. from the Children Cancer Free Foundation (KIKA, project 49). Disclosures: No relevant conflicts of interest to declare.

Description: Background Pediatric classical Hodgkin lymphoma (cHL) is a clonal disorder in an inflammatory background, also known as the microenvironment. This microenvironment is of major importance for growth and survival of the malignant Hodgkin/Reed Sternberg (HRS) cells. HRS cells and the microenvironment communicate through chemo- and cytokines. Blood biomarkers result from this active crosstalk, and may be a surrogate for lymphoma viability (Steidl et al, JCC 2011). Blood biomarkers are important because they hold the promise to be easily available and cost-effective. One promising biomarker in adult patients with cHL is the "Thymus and Activation-Regulated Chemokine, TARC (Plattell et al, Haematologica 2012). Elevated TARC levels are also described in patients with atopic dermatitis (Hijnen et al, J All Clin Immunol 2004). In adult cHL patients about 85% of patients have significantly elevated levels of TARC in pre-treatment serum or plasma compared to healthy controls (Plattell et al). So far nothing is known about TARC in pediatric cHL patients. To define its value as a diagnostic marker in pediatric cHL patients, we compared TARC levels of pediatric cHL patients with control patients. This study was IRB-approved and registered under Dutch Trial registry number 6876. Methods After providing informed consent, plasma and serum samples were collected of newly diagnosed cHL patients before start of treatment. To define normal values of TARC in children, samples were collected from non-cHL randomly selected patients from the hematology, endocrinology and oncology outpatient clinic. Baseline characteristics including history of atopic dermatitis were collected. These control patients were divided in three age groups (0-9,10-14 and 15-18 years). TARC levels were measured by enzyme-linked immunosorbent assay (R&D systems, Human CCL17/TARC Quantikine ELISA Kit). TARC levels of the cHL patients were compared to the control group to obtain ROC curves and calculate the AUC, cross-validated sensitivity and specificity and accuracy of TARC as a diagnostic marker. We hypothesized that pediatric cHL patients had elevated pretreatment TARC levels in both serum and plasma. Analyses were done using SAS V9.4. Results Fourteen cHL patients were included with a median age of 14 (range 11-17) years. Ten (71.4%) were female. Eighty patients were included in the control group with a median age of 12 (range 10 months-18) years. Twenty-nine patients (36.3%) were included in age group 0-9, 25 (31.2%) in age group 10-14 and 26 (32.5%) in age group 15-18. Thirty-nine (48.8%) were female. Patients of the control group had a median TARC value of 71 (range 18-762) pg/ml for plasma and 318 (range 27-1300) pg/ml for serum. TARC plasma and serum levels decreased with age (Spearman correlation -0.26, 2-tailed p=0.0204), but there were no statistically significant pairwise comparisons found between the pre-specified age groups. In the eight control patients (10%) with atopic dermatitis no significantly higher plasma and serum levels were found (plasma median with eczema 97 versus 70 pg/ml without eczema (p=0.71) and serum median with eczema 643 versus 317 pg/ml (p=0.71)). Plasma was collected in 14 cHL patients, and all had elevated TARC levels, with a median plasma level of 18449 (range 1635-55821) pg/mL. Serum samples were collected in 8/14 cHL patients and all had elevated serum TARC levels. Median serum level: 46703 (range 12817-149739) pg/ml. The plasma TARC levels of cHL patients were significantly higher than those of the control group patients (p

Description: Prognosis of refractory and relapsed ALL is poor and improvement requires the introduction of agents with a new mechanism of action. Bortezomib (BTZ) as a proteasome inhibitor is such an agent, and was safe as single agent in phase I studies in children (Blaney 2004; Horton 2007). Messinger et al. (2012) reported a single-arm study that BTZ can be combined safely with conventional drugs; the combination was remarkably effective. BTZ results in sensitization of malignant cells to anticancer agents, both in vitro for leukemias as well as for multiple myeloma patients. In patients with ALL, this effect regarding glucocorticoids has not been addressed yet. It also has not been studied whether BTZ reaches the cerebrospinal fluid (CSF), which is relevant in pediatric leukemias in view of the frequent leptomeningeal involvement. In the setting of the lack of clinical experience with BTZ in children in Europe, we developed a European multicentre feasibility/phase II study in refractory or relapsed ALL, in which all patients get BTZ (NTR 1881, EUDRaCT 2009-014037-25, ITCC 021). A randomisation is done for BTZ to start “early”, on day 1 of treatment, or “late”, on day 8 of treatment. Bortezomib is given as iv push for 4 doses at 1.3 mg/m2/dose, thus in group “early” on days 1, 4, 8 and 11 and in group “late” on days 8, 11, 15 and 18. In addition, all patients receive dexamethasone (10 mg/m2/day in 3 doses for 2 weeks, orally or iv) and vincristine (1.5 mg/m2/dose with a maximum of 2 mg as 1-hour infusion on days 8 and 15), and one intrathecal administration of methotrexate (dose age-adjusted) on day 1. Eligible patients have 2nd or greater relapsed ALL, 1st relapsed ALL after allogeneic stem cell transplantation (allo-SCT) in 1st complete remission (CR1), or refractory 1st relapsed ALL, bone marrow involvement and at least 100 leukemic cells per ul blood. Exclusion criteria includes symptomatic CNS leukemia, among other factors. It is planned to have 24 fully evaluable patients. This interim analysis is limited to a description of pharmacokinetic (PK) data, especially concerning the CSF. As per June 1, 2013 a total of 14 patients has been enrolled, 9 boys and 5 girls, median 8.7 years of age (range, 1.6-16.2). Most had 2nd relapsed ALL (n=9), others 1st relapsed ALL following allo-SCT in CR1 (n=3) or refractory 1st relapsed ALL (n=2). Regarding PK in the peripheral blood, there was remarkable intra- and inter-individual variability in peak plasma concentrations, between patients ranging from 4.7 to 2920 ng/ml 15 minutes after the first administration of BTZ, median 12.4 ng/ml (18.7 ng/ml in the group with BTZ “early”, 12.1 ng/ml in the group with BTZ “late”). Peak levels after the fourth administration were higher, median 41.1 ng/ml (29.5 ng/ml in the group with BTZ “early” and 158.9 ng/ml in the group with BTZ “late”). There was a 10-fold interindividual variation in the area-under the concentration versus time curve until 72 h (AUC[0-72h]) after administration. Median ratio of AUC[0-72h] fourth dose / AUC[0-72h] first dose was 2.7 (range 0.9 – 9.3), which is indicative of accumulation. In all patients, PK of BTZ was studied in the CSF 15 minutes after administration of the first and third dose (group “early” only) of BTZ, as well as 4 days (group “late”) or one week (group “early”) after the last administration. In general, no BTZ was detected with a lower detection limit of 0.1 ng/ml. In 4 patients some BTZ was detected in CSF, at 0.2 – 0.4 – 1.7 - 5.2 ng/ml. Of potential interest, the latter patient also had the highest peak plasma level of BTZ (2920 ng/ml) and the highest AUC. Future analyses in the complete cohort of 24 randomised patients will focus on more extensive population PK and pharmacodynamic analyses, as well as on efficacy of BTZ. The higher peak plasma levels after the fourth dose suggest decreased clearance (especially in the group which received bortezomib “late”), which indeed has been reported in adults and which requires careful monitoring of toxicity over time. Mean peak plasma concentrations in adults were reported to be 173 ng/ml, and thus seem higher. Meanwhile, BTZ does not or hardly penetrate the cerebrospinal fluid and is unlikely to be a drug that significantly adds to the treatment of leptomeningeal involvement in leukemia. Financially supported by the Dutch Foundation Children Cancer-free. Disclosures: Off Label Use: bortezomib in pediatric relapsed acute lymphoblastic leukemia.

Description: Abstract 2038 Poster Board II-15 Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with elevated deoxyguanosine (dGuo) plasma levels. DGuo is converted into dGTP inducing apoptosis in T-cells and this provides the rationale for the development of deoxyguanosine analogues as a potential treatment option for T-cell malignancies. Forodesine (BCX-1777; BioCryst-Mundipharma) is an efficient blocker of PNP activity, thereby boosting the conversion of dGuo into dGTP and raising intracellular dGTP levels. AraG (9-b-D-arabinofuranosyl-guanine) is a compound that is resistant to PNP-mediated degradation that is efficiently converted into AraGTP. AraGTP becomes incorporated in the DNA, blocking DNA synthesis and promoting apoptosis. In a phase II clinical trial, the AraG prodrug Nelarabine enforced a complete remission rate of 55% for pediatric T-ALL patients at 1st relapse. (Berg, JCO 2005). Clinical data of Forodesine treatment in pediatric ALL patients are not yet available. As tested on primary pediatric acute lymphoblastic leukemia (ALL) patient samples (4 T-ALL, 2 BCP-ALL), 1μM of Forodesine is sufficient to completely block PNP and abolish rapid dGuo degradation resulting in a median 7.9 (range 0.5-378) fold raise of intracellular dGTP levels. Accumulation of dGTP is comparable for T-ALL (n=31) and BCP-ALL (n=11) patient samples. This reflects equal intrinsic ability of salvage nucleotide synthesis for both T-ALL and BCP-ALL cells. Cytotoxic effect of Forodesine was tested on primary leukemia cells from newly diagnosed pediatric ALL patients in-vitro by incubating cells with Forodesine (1μM) in the presence of increasing concentrations of dGuo (0.001-50μM). In accordance with selective T-cell toxicity, T-ALL cells were more sensitive to Forodesine/dGuo treatment (median T-ALL LC50 value: 1.1μM dGuo/1μM Forodesine, n=27, p=0.001) compared to BCP-ALL cells, which had a median LC50 value of 8.8μM dGuo/1μM Forodesine (n=30). All patients that responded demonstrated dGTP accumulation (1.5-222.1 fold), although the raise of dGTP levels did not correlate with Forodesine/dGuo toxicity (r2= 0.10, p=0.22). Studying in-vitro responsiveness to AraG, T-ALL cells were more sensitive compared to BCP-ALL cells (p=0.0002) with a median AraG LC50 value of 20.5μM for T-ALL samples (n=24) versus 48.3μM for BCP-ALL samples (n=20). Remarkably, TELAML1 positive BCP-ALL cases were insensitive to AraG treatment (median LC50 value 〉50μM, n=9). No correlation was identified between in-vitro Forodesine/dGuo and AraG cytotoxicities (r2=0.05, p=0.29). Most patient samples that displayed AraG resistance still responded to Forodesine/dGuo treatment. This may be explained by the fact that the uptake of both drugs may be facilitated by different transporters. Using RQ-PCR we could demonstrate that AraG toxicity, in contrast to Forodesine, was significantly associated with ENT1 (equilibrative nucleoside transporter 1) expression levels (p=0.008), which was previously identified as strong predictor for AraC cytotoxicity in pediatric ALL (Stam RW. et al., Blood 2003). AraG cytotoxicity strongly correlated with AraC cytotoxicity (r2=0.71, p

Description: To identify potential regulators of propagation and self-renewal of Acute Lymphoblastic Leukaemia (ALL), we performed an explorative genome-wide RNAi screen followed by CRISPR ex vivo and in vivo validation screens in the t(4;11)-positive ALL cell line SEM. These screens identified the splicing factor PHF5A as a crucial component of the leukemic program. PHF5A is a subunit of the SF3b protein complex, which directs alternative splicing by binding to the branchpoint of pre-mRNA. Mutations in members of this complex including SF3B1 have been implicated in several haematological malignancies. Functional perturbation experiments demonstrated that PHF5A depletion impairs proliferation, viability and clonogenicity in a range of ALL and AML cell lines strongly suggesting that PHF5A is required for leukemic propagation and self-renewal. To identify genetic programs affected by PHF5A inhibition, we performed RNA-seq followed by analysis of differential gene expression and splicing events. We identified 473 genes with differential expression upon PHF5A knockdown. In addition, we performed in-depth analysis of splicing patterns by examining both differential exon/intron usage and exon junction formation. These analyses demonstrated that loss of PHF5A affects splicing of more than 2500 genes with exon skipping and intron retention being the most frequent splicing events. In order to identify processes and pathways affected by PHF5A, we performed gene set enrichment analysis (GSEA) on both differential expression and splicing. While gene sets associated with RNA processing including splicing, turnover and translation were enriched in both data sets, the differential gene expression signature was also linked to DNA repair processes including base excision, mismatch and homologous recombination repair. In line with these findings, knockdown of either PHF5A or its partner protein SF3B1 induced DNA strand breaks as indicated by comet assay and increased y-H2AX levels. Furthermore, both PHF5A and SF3B1 depletion sensitized ALL cells towards the DNA crosslinking agent mitomycin C. Closer inspection of RNA-seq datasets revealed reduced FANCD2 expression and skipping of exon 22 associated with impaired mono-ubiquitination of the FANCD2 protein as a consequence of PHF5A and SF3B1 knockdown. Furthermore, expression of RAD51, a key component of double strand break repair, also decreased upon PHF5A and SF3B1 knockdown. Notably, in vitro pharmacological inhibition of SF3b complex activity using H3B-8800 (or Pladienolide B) showed a very similar effect on FANCD2 expression, and ubiquitination as well as decrease of RAD51 and an increase in y-H2AX levels on a dose and time-dependent manner. This strongly suggests a mechanistic link between impaired RNA splicing and the repair of DNA double-strand breaks. These combined data show that leukemic cells are highly dependent on a functional SF3b splicing complex. Interference with its function results in DNA damage and also sensitizes towards DNA damaging agents pointing towards a possible benefit of the combined application of inhibitors targeting the SF3b complex with more conventional chemotherapy. Disclosures Ponthan: Epistem Ltd: Employment. Zwaan:Sanofi: Consultancy; Incyte: Consultancy; BMS: Research Funding; Roche: Consultancy; Janssen: Consultancy; Daiichi Sankyo: Consultancy; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Pfizer: Research Funding; Celgene: Consultancy, Research Funding. Vormoor:Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.

Description: INTRODUCTION Down syndrome patients with acute lymphoblastic leukemia (DS ALL) are less likely to have a favorable (cyto)genetic subtype and are at higher risk of relapse and treatment-related mortality (TRM) than non-DS ALL (Buitenkamp et al., Blood 2014). At present, the independent predictive value of minimal residual disease (MRD) is unclear and may be biased by an unequal distribution of (cyto)genetic risk groups among DS ALL and non-DS ALL patients. This study was aimed to decipher the prognostic implications of MRD and IKZF1 deletions and the frequency of TRM in a matched cohort of DS ALL cases and non-DS ALL controls. METHODS Each DS ALL patient was matched to 3 non-DS ALL patients based on treatment protocol, induction treatment, cytogenetic subtype, IKZF1 status, age (cutoff at 10 years), and white blood cell count (cutoff at 50,000 cells/µl). For MRD analysis, matching was only on induction treatment and excluded the MRD-guided treatment arm; for survival analyses, matching included the MRD-guided treatment arm, thus resulting in two separately matched cohorts. Patients who died during induction were excluded from matching. Absolute MRD levels were measured with RQ-PCR, log-transformed and analyzed with a multilevel mixed-effects linear regression model. Matched Cox proportional hazard regression models were used to analyze event-free survival (EFS), overall-survival (OS), relapse-free survival (RFS) and mortality in remission as surrogate for TRM. RESULTS Patients treated between 2002 and 2018 on Dutch DCOG-ALL10/11 trials, Australian ANZCHOG-ALL8 and AIEOP-BFM-ALL2009 trials, and UKALL2003 trial were included, resulting in 160 DS ALL and 5313 non-DS ALL patients. Out of these 160 DS ALL patients, 13 died during induction versus 42/5313 non-DS ALL patients (8.1% versus 0.8%, p3, 95% CI=1-12, p=0.05). The effect of IKZF1 deletion was stronger in DS ALL patients than in the non-DS ALL patients in our cohort. CONCLUSION The MRD levels did not differ between DS ALL and non-DS ALL patients when matched for (cyto)genetics and other risk factors. In accordance, the overall relapse rate of DS ALL patients did not differ from that of matched non-DS ALL patients. Similar to non-DS ALL, IKZF1 deletion is an adverse risk factor for DS-ALL, indicating the need for treatment aimed at reducing the high relapse risk. DS ALL patients suffer more frequently from death in induction and from treatment while in remission, which jeopardizes treatment intensification. Therefore, the efficacy of targeted, less toxic therapies such as immunotherapies should be assessed in DS ALL. Disclosures van der Velden: Jansen: Research Funding; BD Biosciences: Research Funding; Amgen: Honoraria, Research Funding. Pieters:jazz farmaceuticals: Consultancy; medac: Consultancy. Zwaan:Celgene: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding; Janssen: Consultancy; Servier: Consultancy; Roche: Consultancy; Incyte: Consultancy; Sanofi: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Daiichi Sankyo: Consultancy.

Description: The homeobox (HOX) genes are a highly conserved family of transcription factors involved in embryonic patterning as well as adult hematopoiesis. Dysregulation of HOX genes, in particular upregulation of HOXA cluster genes, is a frequent event in Acute Myelogenous Leukemia (AML). Recently, we performed a detailed genomic analysis on pediatric non-Down Syndrome Acute Megakaryoblastic Leukemia (non-DS-AMKL) and identified novel fusions involving a HOX cluster gene in 14.9% of the cases. While most fusions were predicted to lead to an in-frame functional protein, several fusions included a non-coding HOX antisense gene (PLEK-HOXA11-AS, C8orf76-HOXA11-AS, HOXA10-AS-CD164) that were predicted to result in a loss of function of these regulatory transcripts. The functional consequence of these events, however, remain unknown. HOXA11-AS (human) and Hoxa11os (mouse) have been previously shown to have mutually exclusive expression with the Hoxa11 transcript throughout development. We therefore hypothesized that loss of function of non-coding HOX antisense genes as a result of these structural variations would cause upregulation of nearby coding HOXA genes that in turn promote leukemogenesis. To test this hypothesis, using CRISPR-Cas9 technology, we genome edited the human AMKL cell line CMK to carry the PLEK-HOXA11-AS translocation. qRT-PCR of HOXA11-AS and HOXA9-11 transcripts in this cell line recapitulated the pattern seen in patient specimens. Specifically, HOXA11-AS expression was significantly diminished while HOXA10 and HOXA11 transcripts were upregulated 1.8-2.5-fold when compared to parental CMK cells (p=0.0385 and p=0.006 respectively). To further investigate the loss of HOXA11-ASin vivo a CRISPR-Cas9 Hoxa11os knockout mouse model was established. qRT-PCR on bone marrow confirmed the loss of Hoxa11os transcripts in heterozygous (Hoxa11os1+/-) and homozygous (Hoxa11os-/-) mice of both genders (p=