Supplementary Table 1 from Epigenetic Modification of CCAAT/Enhancer Binding Protein α Expression in Acute Myeloid Leukemia

KMT2A partial tandem duplication occurs in approximately 5–10% of patients with acute myeloid leukemia and is associated with adverse prognosis. KMT2A wild type is epigenetically silenced in KMT2A partial tandem duplication; re-expression can be induced with DNA methyltransferase and/or histone deacetylase inhibitors in vitro, sensitizing myeloid blasts to chemotherapy. We hypothesized that epigenetic silencing of KMT2A wildtype contributes to KMT2A partial tandem duplication-associated leukemogenesis and pharmacologic re-expression activates apoptotic mechanisms important for chemoresponse. We developed a regimen for this unique molecular subset, but due to relatively low frequency of KMT2A partial tandem duplication, this dose finding study was conducted in relapsed/refractory disease regardless of molecular subtype. Seventeen adults (< age 60) with relapsed/refractory acute myeloid leukemia were treated on study. Patients received decitabine 20 milligrams/meter2 daily on days 1–10 and vorinostat 400 milligrams daily on days 5–10. Cytarabine was dose-escalated from 1.5 grams/meter2 every 12 hours to 3 grams/meter2 every 12 hours on days 12, 14 and 16. Two patients experienced dose limiting toxicities at dose level 1 due to prolonged myelosuppression. However, as both patients achieved complete remission after Day 42, the protocol was amended to adjust the definition of hematologic dose limiting toxicity. No further dose limiting toxicities were found. Six of 17 patients achieved complete remission including 2 of 4 patients with KMT2A partial tandem duplication. Combination therapy with decitabine, vorinostat and cytarabine was tolerated in younger relapsed/refractory acute myeloid leukemia and should be explored further focusing on the KMT2A partial tandem duplication subset. (clinicaltrials.gov identifier 01130506).

Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

Background: MLL partial tandem duplication ( MLL PTD) occurs in approximately 5-8% of patients with acute myeloid leukemia (AML) and is associated with an adverse prognosis. Our group has published that the MLL wild type (WT) allele is epigenetically silenced in MLL PTD; we showed that re-expression of this gene can be induced with methyltransferase (DNMT) and/or histone deacetylase (HDAC) inhibitors. Further, re-expression of MLL WT following combined decitabine and HDAC inhibitor treatment sensitized MLL PTD myeloid leukemia cells to chemotherapy in vitro . We hypothesized that epigenetic silencing of the MLL WT contributes to MLL PTD-associated leukemogenesis and that its pharmacologic re-expression with DNMT and HDAC inhibitors would activate apoptotic mechanisms important for chemo-response in the clinic. We aimed to develop a regimen to be tested in this unique molecular subset of disease. Because of the relatively low frequency of MLL PTD AML, this dose finding study was conducted in relapsed/refractory (R/R) AML regardless of molecular subtype but was enriched for MLL PTD. Methods:In this phase 1 study, adults aged 18-59 years with R/R AML, ECOG 0-2, and preserved organ function were enrolled. Patients received decitabine 20mg/m 2 daily on days 1-10 and vorinostat 400mg daily on days 5-10 for all dose levels. Dose-escalated cytarabine was given on days 12, 14, 16 (six doses) according to the following schedule: dose level (DL) 1, 1.5g/m 2 /q12hr; DL 2, 2g/m 2 /q12hr; DL 3, 2.5g/m 2 /q12hr; and DL 4, 3g/m 2 /q12hr. Standard method 3+3 phase I design was used with the primary objective to determine the maximum tolerated dose and define specific toxicities with this combination therapy, in order to ultimately develop a regimen for MLL PTD AML. Results:Seventeen adults with R/R AML and median age of 46 years (range, 21-59 years) enrolled. The median number of prior induction therapies was 2 (range, 1-4). European LeukemiaNet (ELN) genetic risk classification frequencies were: favorable (n=2), intermediate-I (n=3), intermediate-II (n=5), and adverse (n=7), respectively. Four patients had MLL PTD. A total of 6 patients were treated at DL 1 with no non-hematologic dose limiting toxicity (DLT). DL 1 was expanded after two patients experienced prolonged but uncomplicated myelosuppression. Since both patients achieved complete remission (CR) shortly after passing the day 42 DLT cut off for hematologic recovery, the protocol was amended to allow further time for count recovery (up to 56 days). Three patients each were treated on DL 2 and 3; 5 patients were treated on DL 4 with no other DLTs observed. Diarrhea, nausea, fatigue, febrile neutropenia, and elevated ALT were the most common toxicities (any grade, regardless of attribution), occurring in 41%, 29%, 29%, 35%, and 35% of patients, respectively, but none were DLTs. In regards to ≥ Grade 3 toxicities, febrile neutropenia and catheter-related infections were most common at 35% and 24%. Significant mucositis was not observed. DL 4 was the recommended phase 2 dose (RP2D). CR or CR with incomplete count recovery (CRi) was observed in 6/17 patients (35%, 5 with CR). Of 6 responders, all (n=4) patients with abnormal karyotype achieved cytogenetic remission. The median number of prior therapies for patients achieving CR/CRi was 2 (range, 1-3). Four patients subsequently received allogeneic transplantation. Of the four patients known to have MLL PTD mutations, two responded (1 with CR and 1 with CRi). It is interesting to note that the two patients with MLL PTD mutations who did not respond to treatment also had FLT3-ITD mutations, while this mutation was absent in the two responding patients who had MLL PTD. Conclusions: We successfully determined the RP2D for this novel treatment regimen. The regimen had modest toxicities beyond uncomplicated (though prolonged) myelosuppression, and we propose that the study provides a framework for larger efficacy studies for AML patients with the uncommon but biologically distinct molecular feature of MLL PTD. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number U01CA076576. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Also supported by P30 CA016058/CA/NCI. Disclosures No relevant conflicts of interest to declare.

The t(8;21) rearrangement, which creates the AML1-ETO fusion protein, represents the most common chromosomal translocation in acute myeloid leukemia (AML). Clinical data suggest that CBL mutations are a frequent event in t(8;21) AML, but the role of CBL in AML1-ETO-induced leukemia has not been investigated. In this study, we demonstrate that CBL mutations collaborate with AML1-ETO to expand human CD34+ cells both in vitro and in a xenograft model. CBL depletion by shRNA also promotes the growth of AML1-ETO cells, demonstrating the inhibitory function of endogenous CBL in t(8;21) AML. Mechanistically, loss of CBL function confers hyper-responsiveness to thrombopoietin and enhances STAT5/AKT/ERK/Src signaling in AML1-ETO cells. Interestingly, we found the protein tyrosine phosphatase UBASH3B/Sts-1, which is known to inhibit CBL function, is upregulated by AML1-ETO through transcriptional and miR-9-mediated regulation. UBASH3B/Sts-1 depletion induces an aberrant pattern of CBL phosphorylation and impairs proliferation in AML1-ETO cells. The growth inhibition caused by UBASH3B/Sts-1 depletion can be rescued by ectopic expression of CBL mutants, suggesting that UBASH3B/Sts-1 supports the growth of AML1-ETO cells partly through modulation of CBL function. Our study reveals a role of CBL in restricting myeloid proliferation of human AML1-ETO-induced leukemia, and identifies UBASH3B/Sts-1 as a potential target for pharmaceutical intervention.

In acute myeloid leukemia (AML), about 25–30% of patients harbor a constitutively active receptor tyrosine kinase (RTK) FLT3 encoded by a FLT3 allele harboring internal tandem duplication (FLT3-ITD) mutation. The presence of FLT3-ITD correlates with poor prognosis in AML and it makes FLT3 an attractive therapeutic target in AML. Unfortunately, to date small-molecule inhibitors of FLT3 have resulted in only partial and transient clinical responses with residual leukemic blasts resistant to FLT3 inhibitors detected in blood or bone marrow. In this study, we investigated whether the RTK Axl is responsible for resistance of FLT3-ITD+ AML cells to PKC412 and AC220, FLT3 inhibitors currently under clinical trials for FLT3-ITD+ AML patients. Upon treatment with PKC412 or AC220, phosphorylation of Axl was significantly enhanced in the FLT3-ITD+ MV4-11 AML cell line and in primary blasts from a FLT3-ITD+ AML patient. Consistently, a PKC412-resistant AML cell line and PKC412-resistant primary blasts from FLT3-ITD+ AML patients had significantly higher levels of constitutively phosphorylated Axl and total Axl when compared with a PKC412-sensitive AML cell line and PKC412-sensitive primary blasts from FLT3-ITD+ AML patients. We also found that resistance of AML cells against the FLT3 inhibitor PKC412 and AC220 was substantially diminished by the inhibition of Axl via a small-molecule inhibitor TP-0903, a soluble receptor Axl fusion protein Axl-Fc or knockdown of Axl gene expression by shRNA. Collectively, our study suggests that Axl is required for resistance of FLT3-ITD+ AML cells against the FLT3 inhibitor PKC412 and AC220, and that inhibition of Axl activation may overcome resistance to FLT3-targeted therapy in FLT3-ITD+ AML.

7059 Background: LY3012218 (IMC-EB10) is a monoclonal antibody that binds FLT3 with high affinity and prevents binding of the FLT3-ligand and downstream signaling. Preclinical data using LY301228 demonstrates anti-leukemia activity which appears to be mediated via antibody dependent cell-mediated cytotoxicity (ADCC). Methods: We conducted a phase 1 trial to determine the safety, pharmacokinetics, and preliminary efficacy of LY3012218 in patients with relapsed/refractory acute myeloid leukemia (AML). Patients were eligible if they were ≥ 18 years, had a performance status < 3, adequate organ function, a life expectancy of > 3 months, and were not candidates for higher intensity therapy. Exclusion criteria included stem cell transplant within < 3 months, central nervous system leukemia or isolated extramedullary disease. Patients were treated in 4 dose cohorts (5 mg/kg (n = 3), 10 mg/kg (n = 13), 20 mg/kg (n = 3), 30 mg/kg (n = 5)) and received the drug intravenously weekly on days 1, 8, and 15 for cycle 1 and days 1, 8, 15, and 22 for subsequent cycles. Results: 31 patients were screened and 24 patients were treated. 14 patients were male (58.3%), the median age was 70.3 years (range 26-82), the median number of prior therapies was 2 (range 1-6) and 26% had a baseline FLT3 mutation. 12 patients reported 20 serious AEs with the most frequent including: pneumonia (n = 6, 25%), neutropenia (n = 3, 12.5%) and sepsis (n = 2, 8.3%). Dose limiting toxicities of infection, vomiting (10 mg/kg), and increased transaminases (30 mg/kg) were observed, although these were not clearly related to LY3012218. No MTD was defined. The serum trough concentrations for 5 to 30 mg/kg treatments (mean of Cmin: 41.9 to 739 μg/mL) following the third infusion were above the target trough concentration (33 μg/mL) associated with antitumor activity in preclinical models. Twenty one patients were evaluated for response and all were classified as treatment failures. Conclusions: Treatment with LY3012218 appears safe but did not demonstrate clinical activity as a single agent in relapsed/refractory AML. The lack of efficacy may be due to an absence of ADCC with LY3012218 or the presence of ligand-independent activation of signaling pathways down-stream of FLT3. Clinical trial information: NCT00887926.

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

DNMT3B encodes a DNA methyltransferase implicated in aberrant epigenetic changes contributing to leukemogenesis. We tested whether DNMT3B expression, measured by NanoString nCounter assay, associates with outcome, gene and microRNA expression and DNA methylation profiles in 210 older (⩾60 years) adults with primary, cytogenetically normal acute myeloid leukemia (CN-AML). Patients were dichotomized into high versus low expressers using median cut. Outcomes were assessed in the context of known CN-AML prognosticators. Gene and microRNA expression, and DNA methylation profiles were analyzed using microarrays and MethylCap-sequencing, respectively. High DNMT3B expressers had fewer complete remissions (CR; P=0.002) and shorter disease-free (DFS; P=0.02) and overall (OS; P<0.001) survival. In multivariable analyses, high DNMT3B expression remained an independent predictor of lower CR rates (P=0.04) and shorter DFS (P=0.04) and OS (P=0.001). High DNMT3B expression associated with a gene expression profile comprising 363 genes involved in differentiation, proliferation and survival pathways, but with only four differentially expressed microRNAs (miR-133b, miR-148a, miR-122, miR-409-3p) and no differential DNA methylation regions. We conclude that high DNMT3B expression independently associates with adverse outcome in older CN-AML patients. Gene expression analyses suggest that DNMT3B is involved in the modulation of several genes, although the regulatory mechanisms remain to be investigated to devise therapeutic approaches specific for these patients.

Prognostic gene mutations and distinct gene- and microRNA-expression signatures in acute myeloid leukemia with a sole trisomy 8

High BAALC expression associates with poor outcome in cytogenetically normal AML (CN-AML) patients (pts). Recently, microRNA miR-3151 was discovered in intron 1 of BAALC. To evaluate the prognostic significance of miR-3151 expression levels and to gain insight into the biologic and prognostic interplay between miR-3151 and its host, miR-3151 and BAALC expression were measured in pretreatment blood of 179 CN-AML pts. Gene expression profiling (GEP) was performed using microarrays. In multivariable analysis high miR-3151 expression associated with shorter disease-free and overall survival (DFS: P<.001, HR=2.38; OS: P=.009, HR=1.69), while high BAALC expression predicted failure of complete remission and shorter OS (CR: P<.001, OR=0.23; OS: P<.001, HR=1.93). As approximately one third of pts were discordant in expresser status of the two markers, we next investigated their combined impact on outcome endpoints. Pts with both high miR-3151 and BAALC demonstrated the lowest CR rates (50%) and had a shorter DFS and OS (P<.001 for both DFS and OS) than those expressing both markers at low levels (P<.001 for both DFS and OS) or those who exhibited high expression of only one of the markers (DFS: P=.03, OS: P=.01). Next, we performed a canonical pathway analysis of a miR-3151 expression associated GEP. TP53 signaling was the top affected pathway, and TP53 itself is an in silico predicted miR-3151 target. Luciferase assays proved a direct downregulating effect on the transcript from the TP53-3′-UTR by miR-3151 vs. scramble (-40%, P<.001). Forced miR-3151 expression reduced TP53 mRNA and protein compared to scramble (KG1 cells: -70%, P=.007; MV4-11 cells: -86%, P=.002). Thus miR-3151 directly deregulates TP53 in vitro. In a NSG mouse model, both miR-3151 and miR-3151/BAALC mice had a shorter survival compared to scramble mice (miR-3151/BAALC: P=.006; miR-3151: P=.008), indicating increased leukemogenesis by miR-3151. In conclusion, high miR-3151 expression independently associated with worse outcome in older CN-AML pts and impacts on different outcome endpoints than its host gene BAALC. TP53 was validated as a direct target of miR-3151, which may explain the adverse outcome effects and increased leukemogenesis of miR-3151. The described interplay of an intronic miR and its host may have important biologic and therapeutic implications.

Purpose Molecular risk stratification of acute myeloid leukemia (AML) is largely based on genetic markers. However, epigenetic changes, including DNA methylation, deregulate gene expression and may also have prognostic impact. We evaluated the clinical relevance of integrating DNA methylation and genetic information in AML. Methods Next-generation sequencing analysis of methylated DNA identified differentially methylated regions (DMRs) associated with prognostic mutations in older (≥ 60 years) cytogenetically normal (CN) patients with AML (n = 134). Genes with promoter DMRs and expression levels significantly associated with outcome were used to compute a prognostic gene expression weighted summary score that was tested and validated in four independent patient sets (n = 355). Results In the training set, we identified seven genes (CD34, RHOC, SCRN1, F2RL1, FAM92A1, MIR155HG, and VWA8) with promoter DMRs and expression associated with overall survival (OS; P ≤ .001). Each gene had high DMR methylation and lower expression, which were associated with better outcome. A weighted summary expression score of the seven gene expression levels was computed. A low score was associated with a higher complete remission (CR) rate and longer disease-free survival and OS (P < .001 for all end points). This was validated in multivariable models and in two younger (< 60 years) and two older independent sets of patients with CN-AML. Considering the seven genes individually, the fewer the genes with high expression, the better the outcome. Younger and older patients with no genes or one gene with high expression had the best outcomes (CR rate, 94% and 87%, respectively; 3-year OS, 80% and 42%, respectively). Conclusion A seven-gene score encompassing epigenetic and genetic prognostic information identifies novel AML subsets that are meaningful for treatment guidance.

Acute myeloid leukemia (AML) is hypothesized to be sustained by self-renewing leukemia stem cells (LSCs). Recently, gene expression signatures (GES) from functionally defined AML LSC populations were reported, and expression of a 'core enriched' (CE) GES, representing 44 genes activated in LCSs, conferred shorter survival in cytogenetically normal (CN) AML. The prognostic impact of the CE GES in the context of other molecular markers, including gene mutations and microRNA (miR) expression alterations, is unknown and its clinical utility is unclear. We studied associations of the CE GES with known molecular prognosticators, miR expression profiles, and outcomes in 364 well-characterized CN-AML patients. A high CE score (CEhigh) associated with FLT3-internal tandem duplication, WT1 and RUNX1 mutations, wild-type CEBPA and TET2, and high ERG, BAALC and miR-155 expression. CEhigh patients had a lower complete remission (CR) rate (P=0.003) and shorter disease-free (DFS, P<0.001) and overall survival (OS, P<0.001) than CElow patients. These associations persisted in multivariable analyses adjusting for other prognosticators (CR, P=0.02; DFS, P<0.001; and OS, P<0.001). CEhigh status was accompanied by a characteristic miR expression signature. Fifteen miRs were upregulated in both younger and older CEhigh patients, including miRs relevant for stem cell function. Our results support the clinical relevance of LSCs and improve risk stratification in AML.

Approximately 20% to 25% of patients with acute myeloid leukemia (AML) have a constitutively activated FLT3-internal tandem duplication (FLT3-ITD), and these patients exhibit a poor prognosis. Here, we report that Axl, a receptor tyrosine kinase (RTK) overexpressed and constitutively active in human AML, targets the RTK FLT3 in FLT3-ITD(+) AML. Abrogation of Axl activation by soluble Axl chimeric protein (Axl-Fc) or small interfering RNA (siRNA) diminishes constitutive FLT3 phosphorylation in FLT3-ITD(+) AML. In addition, inhibition of Axl activation by Axl-Fc interferes with the physical interaction between Axl and FLT3. We found that Axl-Fc, a pharmacologic Axl inhibitor, or siRNA targeting Axl inhibits cell growth, induces cell-cycle arrest and apoptosis, and relieves a block in myeloid differentiation of FLT3-ITD(+) AML in vitro. Axl-Fc also suppresses the growth of human FLT3-ITD(+) AML in vivo. Collectively, our data suggest that Axl contributes to the pathogenesis of FLT3-ITD(+) AML through, at least in part, positive regulation of constitutive FLT3 activation. This also suggests that Axl should be pursued as a potential target for the treatment of FLT3-ITD(+) AML.

Abstract 2811 MLL partial tandem duplication (MLL-PTD) is found in 5–8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. The molecular and clinical features of MLL-PTD + AML are different from MLL-fusion + AML, although they share similar worse outcomes. Mouse knock-in model of Mll-PTD has been generated to understand its underlining mechanism (Dorrance et al. JCI. 2006). Using this model, we9ve recently reported hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll PTD/WT mice. Their HSPCs showed increased apoptosis and reduced cell number, but they have a proliferative advantage over wild-type HSPCs. Furthermore, the Mll PTD/WT –derived phenotypic ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, Mll PTD/WT HSPCs never develop MDS or s-AML in primary or transplanted recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for transformation (Zhang et al. Blood. 2012). Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in several MDS, s-AML and de novo AML clinical cohorts, which strongly suggest a potential cooperation for transformation between these two mutations. Our previous study has shown that MLL interacts with and stabilizes RUNX1 (Huang et al. Blood. 2011). Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML. We first generated the mice containing one allele of Mll -PTD in a Runx1 +/− background and assessed HSPCs of Mll PTD/wt /Runx1 +/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous ( Mll wt/wt /Runx1 +/− and Mll PTD/wt /Runx1 +/+ ) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LSK and LSK/SLAM + cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT and Mll PTD/wt /Runx1 +/+ controls. DH FLCs also showed dominant expansion in both serial competitive and serial non-competitive BMT assays compared to WT controls. The DH derived phenotypic ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from Mll PTD/wt /Runx1 +/+ mice. However, DH HSPCs didn9t develop MDS or s-AML in primary or in serial BMT recipient mice. We further generated Mll PTD/wt /Runx1 Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia 1 month after pI-pC injection, and developed pancytopenia 2–4 months later. All these Mll PTD/wt /Runx1 Δ/Δ mice died of MDS induced complications within 7–8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in Mll PTD/wt /Runx1 Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD + patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Disclosures: No relevant conflicts of interest to declare.

Emerging data demonstrate important roles for the TYRO3/AXL/MERTK receptor tyrosine kinase (TAM RTK) family in diverse cancers. We investigated the prognostic relevance of GAS6 expression, encoding the common TAM RTK ligand, in 270 adults (n=71 aged<60 years; n=199 aged ⩾60 years) with de novo cytogenetically normal acute myeloid leukemia (CN-AML). Patients expressing GAS6 (GAS6+), especially those aged ⩾60 years, more often failed to achieve a complete remission (CR). In all patients, GAS6+ patients had shorter disease-free (DFS) and overall (OS) survival than patients without GAS6 expression (GAS6−). After adjusting for other prognostic markers, GAS6+ predicted CR failure (P=0.02), shorter DFS (P=0.004) and OS (P=0.04). To gain further biological insights, we derived a GAS6-associated gene-expression signature (P<0.001) that in GAS6+ patients included overexpressed BAALC and MN1, known to confer adverse prognosis in CN-AML, and overexpressed CXCL12, encoding stromal cell-derived factor, and its receptor genes, chemokine (C-X-C motif) receptor 4 (CXCR4) and CXCR7. This study reports for the first time that GAS6 expression is an adverse prognostic marker in CN-AML. Although GAS6 decoy receptors are not yet available in the clinic for GAS6+ CN-AML therapy, potential alternative therapies targeting GAS6+-associated pathways, for example, CXCR4 antagonists, may be considered for GAS6+ patients to sensitize them to chemotherapy.

It has become clear that there are distinct interrelationships between recurring genetic mutations in acute myeloid leukemia (AML). Whereas certain mutations frequently co-exist in AML, others are mutually exclusive. Mutations in NPM1 and RUNX1 , two of the most commonly mutated genes in AML, are

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

Abstract Cytogenetically normal acute myeloid leukemia (CN AML) patients with a MLL PTD have a poor prognosis compared to CN AML patients with MLL WT. We previously reported that the MLL WT gene in MLL PTD+ CN AML is epigenetically silenced. To investigate in vivo significance of the MLL PTD in the absence of MLL WT on hematopoiesis and leukemogenesis, we generated homozygous MllPTD/PTD and hemizygous MllPTD/- mice. These mice died in utero or as neonates, respectively, precluding further study on adult hematopioesis and leukemogenesis. In the current study, we crossed MllPTD/WT mice with Mll-conditional knock-out (cKO) animals to produce MllPTD/cKO mice. Like the MllPTD/WT mice, the MllPTD/cKO mice survive to adulthood, as both models express Mll WT and PTD. As previously reported by Jude et al, (Cell Stem Cell, 2007) Cre activation in hematopoietic cells of mice carrying the Mll cKO allele resulted in an intragenic deletion in Mll (deltaN allele) that when expressed, the protein was unable to translocate to the nucleus and thereby unable to exert normal Mll function. DNA PCR confirmed the correct MllPTD/deltaN genotype was generated. MlldeltaN/deltaN mice develop bone marrow (BM) failure (marked hypocellularity) at a median of 18 days post-Cre activation. Comparatively, MllPTD/deltaN mice (n=5) survived beyond 52 weeks post-Cre activation (P<0.001), without evidence of BM failure. Also, at one year, MllPTD/deltaN animals exhibit equivalent white blood cell counts and hematocrit when compared to MllWT/WT, MllPTD/WT and MllPTD/cKO controls. To evaluate whether Mll PTD function was maintained in bone marrow, qRT-PCR analysis of the Mll transcriptional target Hoxa9 at 12 days post-Cre activation revealed a 20-fold reduction in Hoxa9 mRNA levels in MlldeltaN/deltaN animals and ∼4-5 fold increase in MllPTD/deltaN and MllPTD/cKO BM (P=0.025) compared to WT BM. MllPTD/cKO animals, with two functional copies of Mll, had nearly equivalent overexpression of HoxA9 as seen in MllPTD/deltaN mice that maintain only one functional copy of Mll, supporting our previous hypothesis that the Mll PTD acts as a gain-of-function mutation. In conclusion, these data demonstrate for the first time that Mll PTD alone can support adult hematopoiesis. In conclusion, these data demonstrate for the first time that Mll PTD alone can support adult hematopoiesis and even though Hoxa9 is upregulated, another oncogene is required for leukemogenesis. Absence of Mll WT is not sufficient to promote acute leukemogenesis in Mll PTD+ mice, but appears to be a contributory factor. We recently reported that MllPTD/WT mice crossed with Flt3ITD/WT mice, the latter of which also do not develop AML (Lee et al. Cancer Cell, 2006), generated double mutant mice that do develop AML. Leukemic blasts from these mice exhibit a reduction in Mll WT expression (Zorko et al, Blood, 2012), recapitulating what is seen in human MLL PTD+ CN AML. Citation Format: Nicholas A. Zorko, Daniel A. Yanes, W. Courtland Lewis, Daniel L. Brook, Kelsie M. Bernot, Ronald F. Siebenaler, Susan P. Whitman, Elshafa H. Ahmed, Kathleen K. McConnell, John Nemer, Patricia Ernst, Gang Huang, Guido Marcucci, Michael A. Caligiuri. The partial tandem duplication of Mll (Mll PTD) is a gain-of-function in the absence of Mll wildtype (Mll WT) in adult mouse hematopoiesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3849. doi:10.1158/1538-7445.AM2013-3849