Biology Of Acute Myeloid Leukemia Research Articles

Harnessing the Immune System Against Leukemia: Monoclonal Antibodies and Checkpoint Strategies for AML.

Acute myeloid leukemia (AML) is the most common leukemia among adults and is associated with a poor prognosis, especially in patients with adverse prognostic factors, older age, or relapsed disease. The last decade has seen a surge in successful immune-based therapies in various solid tumors; however, the role of immune therapies in AML remains poorly defined. This chapter describes the rationale, clinical data, and toxicity profiles of immune-based therapeutic modalities in AML including naked and conjugated monoclonal antibodies, bispecific T-cell engager antibodies, chimeric antigen receptor (CAR)-T cells, and checkpoint blockade via blockade of PD1/PDL1 or CTLA4. Monoclonal antibodies commonly used in AML therapy target highly expressed "leukemia" surface antigens and include (1) naked antibodies against common myeloid markers such as anti-CD33 (e.g., lintuzumab), (2) antibody-drug conjugates linked to either, (a) a highly potent toxin such as calicheamicin, pyrrolobenzodiazepine, maytansine, or others in various anti-CD33 (gemtuzumab ozogamicin, SGN 33A), anti-123 (SL-401), and anti-CD56 (lorvotuzumab mertansine) formulations, or (b) radioactive particles, such as 131I, 213Bi, or 225Ac-labeled anti-CD33 or CD45 antibodies. Novel monoclonal antibodies that recruit and promote proximity-induced cytotoxicity of tumor cells by T cells (bispecific T-cell engager [BiTE] such as anti CD33/CD3, e.g., AMG 330) or block immune checkpoint pathways such as CTLA4 (e.g., ipilimumab) or PD1/PD-L1 (e.g., nivolumab) unleashing the patients T cells to fight leukemic cells are being evaluated in clinical trials in patients with AML. The numerous ongoing clinical trials with immunotherapies in AML will improve our understanding of the biology of AML and allow us to determine the best approaches to immunotherapy in AML.

Expression of DDIT4 Is Correlated with NOTCH1 and High Molecular Risk in Acute Myeloid Leukemias

In a meta-analysis, our team identified previously that DDIT4 expression is related with a worse outcome of several cancer types, including acute myeloid leukemia (AML). DDIT4 gene product is a repressor of mTOR activity and recent findings in the triple negative breast cancer model suggest that mTOR pathway inhibition lead to the enrichment of cancer stem cells, explaining indirectly the relationship between DDIT4 and the outcome and suggesting that a high DDIT4 expression could be related with expression of stem-cells markers, such as NOTCH1.We evaluated 200 AML patients from the TCGA dataset. Clinical features and level 2 data of gene expression were retrieved from the website cbioportal.org for mTOR pathway and stem cell markers including ALDH genes. Gene Set Enrichment Analysis (GSEA) for biological processes was done comparing patients with high risk vs low risk determined by molecular abnormalities.GSEA analysis found not differences in the mTOR pathway. DDIT4 is not included in none gene ontology datasets. NOTCH1 and DDIT4 expression was enriched in patients with high molecular risk and statistical differences were found mainly between good and poor prognostic groups (score -0.45 and -0.46, respectively). We next compared the correlation between DDIT4 and NOTCH1 in the entire cohort. The correlation was significant (R2=0.121; P<0.0001). NOTCH1 was over expressed in the M0 FAB subtype while the lowest expression was found in M3, M4, M5 and M7 subtypes.This work shows the relevance of DDIT4 and NOTCH1 in the biology of AML and his association with most undifferentiated subtypes. Association between DDIT4 with NOTCH1 should be evaluated with the intention of explore therapeutic opportunities. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Cell of Origin in NPM1 Positive Acute Myeloid Leukemia (AML) Is Prognostic

Abstract Mutations in the nucleophosmingene (NPM1) are common in patients with cytogenetically-normal acute myeloid leukemia (AML) and have been associated with positive outcomes. Although most leukemic stem cells (LSCs) arise from CD34+ primitive progenitors, our recent data suggest that a fraction arise from less primitive CD34- progenitors and seem to have a better prognosis (Gerber et al. Haematologica 2016). Conversely, poor-risk AMLs such as those harboring internal tandem duplications of the FMS-like tyrosine kinase 3 (FLT3-ITD) or with poor-risk cytogenetics appear to arise from LSCs with a primitive hematopoietic stem cell (HSC) phenotype. NPM1-mutated AMLs can be either CD34- or CD34+, and have been associated with variable prognoses. We hypothesize that the cell of origin, CD34- or CD34+, of NPM1-mutated AML has prognostic implications. A retrospective cohort analysis was conducted using electronic medical record data from patients aged 18 or older with newly-diagnosed NPM1-mutated AML treated with intensive induction chemotherapy at Johns Hopkins Hospital from 2008-2015. NPM1 and FLT3-ITD mutations were determined from PCR assays on diagnostic samples. We (Gerber et al. Haematologica2016) and others have shown that CD34+ cells represent normal, non-leukemia progenitors when they comprise &lt;1% of mononuclear cells (MNCs); thus, &gt;/= 1% MNCs was the cut-off used to distinguish CD34+ from CD34- AMLs. Clinical outcomes including complete remission (CR) rate were compared using Chi-squared tests and event-free survival (EFS) and overall survival (OS) were estimated per the Kaplan-Meier method, using Wilcoxon tests to determine the impact of CD34 status. Of 46 consecutive, newly diagnosed NPM1-positive patients treated between 2008-15, 36 (78%) had normal cytogenetics and 37 (80%) had de novo AML. 26/46 (57%) had CD34+ disease; of these, 18/26 (69%) had FLT3-ITD mutations, compared to 9/20 (45%) with CD34- AML (p=0.10). In the CD34- patients with FLT3-ITD, the mean allelic ratio (mutant to wild type allele) ±SEM was 0.26±.08 compared to 2.3±1.0 in the CD34+ patients (p=0.07). Comparing CD34+ and CD34- patients, there were no significant differences in mean age at diagnosis (52 vs 59), sex, initial hemoglobin, platelet count, or lactate dehydrogenase, but CD34+ patients had significantly higher baseline white blood cell count (mean 78 vs 38 x 103 cells/mm3; p=0.04). There was a trend toward lower CR rates following induction for CD34+ patients (69 vs 89%; p=0.11), and similar proportions of patients underwent bone marrow transplantation (BMT) (50% vs 37%, p=0.38). However, CD34+ patients had significantly shorter EFS (figure 1; median 8.0 vs 34.3 months; p=0.04), and a trend toward inferior OS (15.4 vs 35.4 months; p=0.36). The lack of statistical significance in OS may reflect effective salvage by allogeneic transplantation. In conclusion, in this small retrospective cohort analysis of NPM1-mutated AML patients, CD34 expression was associated with significantly reduced EFS and, among FLT3-ITD mutated patients, a trend toward higher allelic ratios. Hence, CD34 positivity may be an early indicator of poor prognosis in NPM1+ AML and requires further characterization to determine its full prognostic and predictive value in guiding AML therapy. These data support evidence that the biology of AML is not only a function of genetic mutations, but also in part a functionof the stage of hematopoietic differentiation at which the leukemogenic mutations develop. Figure 1 EFS of NPM1-mutated AML patients by CD34 expression. Median follow-up of 23 months. EFS was significantly shorter for CD34+ patients (median 8.0 vs 34.3 months; p=0.01). Figure 1. EFS of NPM1-mutated AML patients by CD34 expression. Median follow-up of 23 months. EFS was significantly shorter for CD34+ patients (median 8.0 vs 34.3 months; p=0.01). Disclosures No relevant conflicts of interest to declare.

Targeting MAPK Signaling Pathway with Cobimetinib (GDC-0973) Enhances Anti-Leukemia Efficacy of Venetoclax (ABT-199/GDC-0199) in Acute Myeloid Leukemia Models

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. Pro-survival molecules of BCL-2 family play critical roles in leukemia transformation and chemoresistance. The anti-leukemia potency of selective BCL-2 inhibitor venetoclax (ABT-199/GDC-0199) has been demonstrated in AML models (Pan et al. Cancer Discovery 2014). However, venetoclax is often associated with resistance due to its poor inhibition of MCL-1. RAF/MEK/ERK (MAPK) pathway is commonly activated in AML, and can stabilize anti-apoptotic MCL-1 and inactivate the pro-apoptotic BIM. In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib).First, anti-leukemia activity of cobimetinib and venetoclax was examined in 18 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death, as compared to the single agent treatment (Fig 1A). Cobimetinib inhibited cell proliferation in the majority of AML cases (34.2 ± 23.7%) and the cell growth suppression was more profound in the combination group (60.2 ± 28.8%, p<0.001) (Fig 1A). The clonogenic potential of myeloid progenitors was significantly suppressed by the combination (82.5 ± 20.0%), as compared to cobimetinib (38.3 ± 14.6%, p=0.01) or venetoclax (41.9 ± 18.6%, p<0.05). The normal progenitor function was minimally affected.We next investigated signaling patterns and BCL-2 family protein expression in AML stem/progenitor cells using a 34-antibody panel for CyTOF. In AML 4400240, we identified 10 distinct subpopulations based on cell surface marker expression that were used to build the SPADE tree. BCL-2 and MCL-1 were both enriched in progenitor populations phenotypically positive for CD34, CD38, CD123 and HLA-DR (A2-5 and A9-10, Fig 1B and 1C). G-CSF-induced p-ERK and SCF-induced p-S6 signaling pathways were efficiently suppressed by cobimetinib (Fig 1D). The inhibition of p-S6 may reflect the sensitivity to cobimetinib of this sample (IC50=1.68 nM); consistent with previous study that suppression of TORC1 predicted response to MEK inhibitor (Corcoran et al. Sci Transl Med 2013).We have previously reported activity of venetoclax/cobimetinib combination in a panel of myeloid leukemia cell lines and revealed distinct response patterns to single agents and combination (Han et al. ASH 2015). Functional proteomics RPPA data indicated that p-ERK, p-S6 and p-RSK pathways were significantly down-regulated in response to the combination in cells showing synergy. Western blotting was performed to validate the RPPA data in 4 cell lines representing different response patterns. Cobimetinib inhibited p-ERK when used at high dose (1 _M) in all cell lines. In turn, inhibition of p-S6 at both ser235/236 and ser240/244 sites occurred at low dose of cobimetinib in sensitive cell lines (OCI-AML3 and MV4-11) and required high dose in resistant lines. In OCI-AML3 cells that showed synergy to combination, elevated levels of cleaved PARP was observed in the combination group, which was likely due to suppression of both BCL2:BIM and MCL-1:BIM complex, followed by release of free BIM to induce cell death.To test the efficacy in vivo, we injected NSGS mice with genetically engineered MOLM3/Luc/GFP cells. Bioluminescent imaging demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the venetoclax (100 mg/kg/d) group and in venetoclax plus cobimetinib (10 mg/kg/d) co-treated mice (Fig 1E and 1F). Human CD45 engraftment and cell counts in both bone marrow and spleen demonstrated a trend towards decreased tumor burden when venetoclax was combined with cobimetinib in vivo (Fig 1G and 1H).In summary, combinatorial blockade of MAPK and BCL-2 pathways promotes cell death and suppresses proliferation in the majority of primary AML cells. The anti-leukemia efficacy of combined blockade of signaling and pro-survival pathways is associated with downregulation of MCL-1, release of pro-death BIM and suppression of p-S6. Additional novel transcriptional biomarkers of response are now being analyzed and will be presented. Combined efficacy of these agents is under clinical investigation in a Phase I trial in elderly relapsed/refractory AML (NCT02670044). [Display omitted] DisclosuresLeverson:AbbVie: Employment, Other: Shareholder in AbbVie. Monique:Genentech: Employment. Phillips:AbbVie Inc.: Employment. Chen:AbbVie: Employment. Jin:AbbVie Inc.: Employment. Daver:Ariad: Research Funding; Sunesis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Kiromic: Research Funding; BMS: Research Funding; Otsuka: Consultancy, Honoraria; Karyopharm: Honoraria, Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Kantarjian:Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. Sampath:Genentech: Employment. Konopleva:AbbVie: Research Funding; Genentech: Research Funding.

Biological Associations and Clinical Impact of Differential Expression of the Pre-Mir-29a/b-1 and Pre-Mir-29b-2/C Clusters in Acute Myeloid Leukemia

Expression levels of miR-29 family members (i.e. miR-29a, miR-29b, &miR-29c) are deregulated in various neoplastic diseases, including acute myeloid leukemia (AML), known to affect DNA-methylation profiles by targeting epigenetic modifiers, & have been shown to be important for normal hematopoietic stem cell function. Mir-29 is organized in two distinctively regulated bi-cistronic clusters: the miR-29a/b-1 cluster & the miR-29b-2/c cluster.Here we evaluated the biological associations & clinical impact of the differential expression of pre-miR-29a/b-1 &pre-miR-29b-2/c clusters in AML.We analysed121 AML patients (pts) (median age 63 years [y], range 37-75 y) who have been consolidated with hematopoietic stem cell transplantation following non-myeloablative conditioning (nma-HCT; Fludarabin 30 mg/m2 on day -4 till -2 & 2 Gy total body irradiation) between 2000 & 2014 with pretreatment bone marrow material (BM) available. Disease status at nma-HCT was first (CR1 62%) or second complete remission (CR2 18%) or CR with incomplete peripheral recovery (CRi 20%). The mutation status (mut) of the ASXL1, CEBPA, DNMT3A IDH1, IDH2, NPM1, &TP53 gene & the FLT3-ITD &EVI1 expressionstatusas well as common surface marker expressions were assessed at diagnosis. European LeukemiaNet (ELN) classification was favorable (25%), intermediate-I (23%), intermediate-II (21%), adverse (27%) or unknown (4%). Pretreatment pre-miR-29a/b-1 &pre-miR-29b-2/c clusters expressionin bone marrow (BM)was measured by quantitative reverse transcription polymerase chain reaction & normalized to 18S. The median normalized gene expression defined high & low pre-miR-29a/b-1 &pre-miR-29b-2/c clusterexpressers. Median follow-up was 4.4y for pts alive.At diagnosis a high pre-miR-29a/b-1 expression did not associate with clinical characteristics. High pre-miR-29a/b-1 expressers were less likely to be TP53 mut (p=.01). Pts with high pre-miR-29b-2/c expression at diagnosis had higher BM blast counts (p=.01), were more likely to have a normal cytogenetics (CN, p=.03) & were less likely to be TP53 (p=.004) or ASXL1 mutated (p=.03).When we combined the expression status information of the two miR-29 clusters we found that AML blasts of pts with high expression of both clusters were less likely to be CD34 (p=.05) or CD117 (p=.04) positive & more likely to be CD11b positive (p=.05). These pts more often had CN-AML (p=.04) & better ELN genetic risk (p=.03). High expressers of both miR-29 clusters were also more likely to be DNMT3A mut (p=.01) & less likely to be EVI1 positive (p=.007). Noteworthy, none of the pts with high expression of both clusters had a TP53 (p=.16) or ASXL1 mutation (p=.08). Pts with a high expression of both miR-29 clustershad a significant longer relapse free survival (RFS, p=.01, Figure 1a) & overall survival (OS, p=.03) compared to pts with low expression of one or both miR-29 clusters.In conclusion, high expression of pre-miR-29a/b-1 &pre-miR-29b-2/c associated with different clinical & genetic characteristic at AML diagnosis. High expressers of both clusters were more often DNMT3A mutated, a gene targeted by miR-29. Furthermore, none of these patients harbored TP53 mutations, a gene known to be indirectly activated by miR-29 family members. These findings provide new insights into the miR-29 associated AML biology, which may contribute to the observed impact on AML pts outcomes. While we observed a trend for better survival for each miR-29 cluster, pts with high expression of the pre-miR-29a/b-1 & the pre-miR-29b-2/c clusterhad significantly longer RFS & OS. [Display omitted] DisclosuresPoenisch:Mundipharma: Research Funding.

Inv(16) AML Engrafts Human Cytokine Knock-in Mice

Favorable-risk acute myeloid leukemia (AML) constitutes up to 40% of newly diagnosed AML cases. However, only 66% of younger and 33% of older patients are alive 3 years post diagnosis. Studying the biology of these entities in vivo has been inherently difficult: in fact, faithful xeno-engraftment of human AML in immunodeficient mice has been limited to higher-risk AML.To address this limitation we hypothesized that a human myelopoiesis-supportive environment may permit xeno-engraftment of favorable-risk AML: we transplanted primary human AML with isolated NPM1 mutation and AML with inv(16) into knock-in (KI) mice with human versions of genes encoding M-CSF, IL-3, GM-CSF and Thrombopoietin. Newborn MISTRG mice (Rongvaux et al. Nat Biotechnol. 2014) were sub-lethally irradiated, intra-hepatically injected with AML blasts and analyzed 16-24 weeks post transplantation.Flow cytometric analysis of bone marrow (BM) and blood of cytokine KI versus NSG mice revealed profound differences: Average engraftment was 36% in the BM of cytokine KI mice compared to only 10% in NSG mice across all patient samples transplanted. In line with engraftment levels, engraftment efficacy was higher in cytokine KI mice. Importantly, average engraftment in cytokine KI mice approached BM and peripheral blast count observed in human AML patients, while NSG engraftment levels were significantly lower. AML engraftment in cytokine KI mice was found to be independent of expression of the SIRPα transgene in mice expressing all four cytokines. NMP1mut AML showed a trend towards higher BM engraftment in MI(S)TRG versus NSG mice. Expression of the NPM1mutallele was more than 3-fold higher in MI(S)TRG mice compared to NSG mice. Performing targeted next generation sequencing in human and engrafted murine BM, we found comparable allelic frequency ranging from 38% to 51% for NPM1 mutation in a patient’s leukemia and engrafted cytokine KI mice. Of note, even the composition of NPM1 mutation subclones was constant between patient and cytokine KI recipients.In contrast to NPM1mut, a striking difference in quantitative inv(16) AML engraftment was observed between cytokine KI and NSG mice, both by aggregate and split-sample analysis. Serial transplantation further validated engraftment of an inv(16) leukemia-initiating cell population in cytokine KI mice. To dissect the contribution of respective single human KI cytokines to inv(16) AML propagation, we analyzed engraftment in Rag2-/-gc-/- mice carrying, in addition to a human SIRPα transgene, further KI genes for human thrombopoietin (STRG), M-CSF and Thrombopoietin (MSTRG), and M-CSF, IL-3, GM-CSF and Thrombopoietin (MI(S)TRG). STRG mice, as NSG mice, did not support engraftment. In contrast, addition of M-CSF in MSTRG mice led to robust engraftment of inv(16) AML, while addition of IL-3 and GM-CSF in MI(S)TRG mice resulted in only marginal, if any, improvement over MSTRG mice. Of note, knock-in of human M-CSF has previously been shown to be sufficiently cross-reactive to rescue the murine M-CSF knock-out phenotype (Rathinam et al. Blood 2011). We thus reasoned that the engraftment advantage observed in MSTRG mice was likely due to human M-CSF effects on blasts and not due to reduction in mouse macrophages. Together, this establishes a central role for M-CSF in maintenance of inv(16) AML.When profiling cytokine receptor expression for TPO, M-CSF, IL-3 and GM-CSF on inv(16) AML blasts in comparison to NPM1mut AML and an aggregate of non-favorable-risk AML cases, using microarray data from an international cohort study (Wouters et al. Blood 2009), inv(16) AML showed significantly higher expression of each of the 4 receptors compared to NPM1mut AML, and significantly higher expression of M-CSFR and GM-CSFR compared to non-favorable-risk AML; the overall highest difference in expression was observed for M-CSFR. Moreover, gene set enrichment analysis demonstrated enrichment of M-CSF-inducible genes in inv(16) AML compared to NPM1mutAML and non-favorable-risk AML cases, confirming increased signaling through the M-CSF pathway.In sum, we here describe the first faithful and highly efficacious xenotransplantation model of favorable-risk, and specifically, inv(16) AML. We further demonstrate a strong dependency of this subtype on M-CSF, a finding that might provide a basis for therapy optimization studies informed by an improved understanding of favorable-risk AML biology. DisclosuresNo relevant conflicts of interest to declare.

The Human Specific microRNA-941 Is a Key Member of a microRNA Signature of Functionally Validated Leukemic Stem Cells from Patients with Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is initiated and maintained by a subpopulation of leukemic stem cells (LSCs), which are defined by their property to serially engraft immunocompromised mice. Based on this a LSC-associated gene expression was identified which was shown to be prognostically relevant in a retrospective analysis (Eppert et al, Nature Medicine 2011). microRNAs (miRs) play important roles in leukemogenesis, serve as potential tumor markers and novel therapeutic targets. In the current analysis we wanted to establish a miRNA profile of functionally validated LSCs from AML patients of various molecular and cytogenetic subgroups by conducting a comprehensive functional genomics study.Method: In total, 17 de novo AML samples were sorted for CD34+ granulocyte-macrophage progenitor (GMP), CD34+ lymphoid-primed multi-potential progenitor (LMPP) and CD34- subpopulations. Sorted cells were transplanted into sublethally irradiated non-obese diabetic/severe combined immunodeficient Gamma (NSG) mice. Those subpopulations that gave rise to human leukemic cell engraftment > 1% were classified as LSCs and sub-divided into LMPP-LSCs and GMP-LSCs and compared to the CD34- cell compartment which did not give rise to human leukemic cell engraftment (non-LSC). In parallel corresponding counterparts were highly purified from healthy bone marrow (BM). miRNA libraries were prepared using the Illumina TruSeqTM Small RNA Sample Preparation Kit and sequenced as single end reads on a Illumina HiSeq 2000. The bioconductor package limma was used for identifying differentially expressed miRNAs, the made4 package for correspondence analysis and between group comparisons. Quantitative RT-PCR validation on bulk and sorted populations (LMPP-LSC, GMP-LSC, CD34-) tested the expression levels of the identified miRNAs.Results: We assessed the engraftment potential of 17 AML samples, sorted into the LMPP, GMP and CD34- fraction. The LMPP or GMP fractions of 8 patients showed a leukemic graft in the NSG mouse model, whereas the CD34- fractions were not able to show a human engraftment in these mice. To the end subpopulations of 8 validated patient samples and corresponding stem-, progenitor, and CD34- subpopulations of normal bone marrow samples (n=3) were analyzed. Global miRNA expression profiles of subpopulations from normal and patient samples were compared using correspondence analysis based between group analysis. First, LSC subpopulations were compared to the leukemic CD34- Non-LSCs: between the LMPP-LSCs and the CD34- non-LSCs 15 miRNAs were differentially expressed in the LMPP-LSCs; between the GMP-LSCs and the CD34- non-LSCs only six miRNAs were found to be differentially expressed. Using these miRNAs, an unsupervised hierarchical clustering showed that the LMPP-LSCs, the GMP-LSCs and leukemic CD34- samples formed non-overlapping clusters. In normal bone marrow, 12 miRs showed differential expression between normal BM LMPP and CD34- population and 36 miRNAs between BM GMP vs. BM CD34- cells.We then compared the miRs that were differentially expressed in the patients compared to the differentially expressed miRs between the normal BM populations: except for one miR (miR-182), the fifteen differentially expressed miRs in the LMPP-LSC versus leukemic CD34- samples did not show overlap with the twelve miR significantly differentially expressed between normal BM LMPP and CD34- cells. When the GMP-LSC to leukemic CD34- Non-LSC comparison was cross-compared to the BM GMP to normal BM CD34- comparison, only two miRs (miR-148a, miR-320a) were unique to the patient samples. When we finally compared the LMPP-LSC specific 14 miR to the 2 GMP-LSC specific miRs, interestingly, we observed that the GMP-LSC miRs were a subset of the LMPP-miRs. Thus, 14 miRs defined a set of miRs differentially expressed in functionally validated AML LSCs. Importantly, 4 of these 14 miRs represent miR-941 isoforms. This miR has not been linked to leukemogenesis yet. It is one of the very few human-specific miRs which developed de novo in the human lineage after separation of the human and the chimpanzee lineages between six and one million years ago and preferentially targets genes in hedgehog- and insulin-signalling pathways. Taken together, combining functional validation of AML LSCs with next generation sequencing allows to identify miRs so far not linked to AML biology and LSC properties. [Display omitted] DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

HIF-1α Promotes Cbfbeta-SMMHC-Induced Acute Myeloid Leukemia Development

Acute myeloid leukemia (AML) develops by the acquisition of genomic alterations which initiate different pathways leading to full-blown malignancy. The type of genomic driver alteration governs AML biology and clinical disease course. Translocations involving the core-binding factor (CBF) complex such as t(8:21) and inv(16) represent distinct subgroups of AML genomic drivers. However, additional factors promoting CBF AML leukemogenesis remain largely unknown. Due to the hypoxic nature of the bone marrow the activation of the ubiquitous hypoxia-sensing pathway via HIF-1a might promote CBF leukemogenesis. Nevertheless, in different AML models HIF proteins were recently considered as oncogenic drivers and also as tumor suppressors. Therefore, we here aimed to clarify the role of HIF-1a in CBF AML.Initially, we investigated the impact of hypoxia-sensing pathway activation on the growth of the CBF AML cell lines Kasumi-1 [t(8:21)] and ME-1 [inv(16)]. Kasumi-1 cells showed increased growth after 24 as well as 48 hours at 3% compared to 21% oxygen conditions. In contrast, ME-1 cellular growth was not altered by hypoxic culture conditions. Next, we genetically modified the HIF-1a pathway in the CBF AML leukemogenesis context with a previously established conditional inv(16) mouse model (Cbfb+/56M). These mice express the Cbfbeta-SMMHC oncoprotein upon Cre-mediated recombination. In preliminary studies we could show neither vav-iCre mediated Hif-1a gain- nor loss-of-function altered steady-state hematopoiesis, which was recently confirmed by others (Milica Vukovic et al., Blood 2016). We proceeded by crossing conditional inv(16) with conditional mice overexpressing a HIF-1a variant (LSL-HIF1dPA) being insensitive to oxygen-dependent degradation. The conditional inv(16) and HIF1dPA alleles were simultaneously activated using the ubiquitous hematopoietic vav-iCre transgene. Strikingly, we observed accelerated leukemia development in inv16;HIF1dPA mice compared to inv(16) mice without genetic HIF-1a pathway alterations (median survival 68 vs. 116 days; Figure 1). In a complementary approach we crossed the vavi-Cre conditional inv(16) system into a conditional Hif-1a knock-out (KO) background [inv(16);Hif-1aKO mice]. Here we observed significantly delayed leukemia onset in inv(16);Hif-1aKO mice compared to inv(16) control mice (median survival 223 vs. 116 days; Figure 1). The gross leukemic phenotype with high peripheral leukocyte count, splenomegaly and liver infiltration did not differ between the groups. Finally, we analyzed deletion of the conditional Hif-1aKO allele of leukemic inv(16);Hif-1aKO mice and observed complete recombination resulting in a Hif-1a null constellation. Thus, AML in inv(16);Hif-1aKO mice did not develop from clones which evaded vav-iCre mediated Hif-1a deletion.In conclusion, HIF-1a represents a critical factor promoting murine inv(16) leukemogenesis and it will be worthwhile studying the impact of genetic or pharmacologic HIF-1a alteration in established inv(16) leukemia. [Display omitted] DisclosuresDührsen:Roche: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Göthert:Proteros: Consultancy; Ariad Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria.

Nuclear NFATc1 Causes Development of Fms-like Receptor Tyrosine Kinase 3 Receptor (FLT3) Internal Tandem Duplication Mutation-Positive (FLT3-ITD) Acute Myeloid Leukemia (AML) and Mediates Drug Resistance

Abstract Background: Although an internal tandem duplication (ITD) of the Fms-like receptor tyrosine kinase 3 receptor (FLT3) confers a very poor prognosis in acute myeloid leukemia (AML), it is insufficient to cause AML, suggesting important cooperating genetic events. NFATc1 is a member of the nuclear factor of activated T-cells (NFAT) transcription factor family, whose activation is dependent on nuclear translocation. NFATc1 is well known for its central role in regulating T-cell development and function, but was also shown to contribute to neoplastic transformation in diverse types of cancers. We have recently demonstrated that NFATc1 is overexpressed in primary FLT3-ITD+ AML causing resistance to the FLT3-kinase inhibitor sorafenib in vitro. Here we asked, whether NFATc1 induces AML transformation and drug resistance when expressed in the context of FLT3-ITD+ in vivo. Methods: Using the stem cell like (SCL)-promoter, Cre-recombinase inducible expression of a constitutively nuclear NFATc1 protein (cnNFATc1) was targeted to the early hematopoietic stem cell compartment. The SCL-Cre/cnNFATc1 mice were subsequently crossed with transgenic FLT3-ITD mice to yield Scl-Cre+/-/cnNFATc1+/+/FLT3-ITD+/+ mice (FCN+/+), which co-express cnNFAT and FLT3-ITD in the stem cell compartment. Results: Whereas FLT3-ITD mice developed a non-lethal, mild myeloproliferative disease, co-expression of FLT3-ITD with cnNFATc1 led to rapidly fatal AML. The median survival of FCN mice was only four months (range, 2 to 8 ). In median, FCN mice had 15-fold (range, 10 to 20) increased white blood cell count, a high proportion of circulating peripheral blasts, severe anemia and thrombocytopenia. There was also a significant B-cell developmental block. As opposed to F+/+ animals, FCN+/+ mice revealed a severe spleno- and hepatomegaly secondary to a gross leukemic infiltration, which disrupted the normal anatomical architecture of the involved organs. Fluorescence activating cell sorting and colony forming unit (CFU) assays demonstrated that cnNFATc1, when expressed in the context of FLT3-ITD, leads to a significant expansion of the lin-Sca+c-kit+ (LSK) stem cell-, progenitor cell (LK)-, the Gr-1-/CD11b+ monocytic and Gr-1low/CD11b+ immature myeloid compartments in bone marrow and spleen. FCN+/+-AML was polyclonal, and re-transplantable in secondary wild-type recipient mice. In vivo, cnNFATc1 caused not only potent FLT3-ITD inhibitor resistance to sorafenib and to the more FLT3-ITD-selective compound quizartinib, but also to chemotherapy. Comparative mRNA-sequencing of sorted LK and LSK compartments in wild-type, F+/+, C+/- N+/+, and FCN+/+ mice will reveal the genetic basis of the biological cooperativity between NFATc1 and FLT3-ITD in stem and progenitor cell compartments. Consistent with the murine data, a data set of AML patients (n=163) revealed that NFATc1 overexpression alone or when combined with FLT3-overexpression was associated with a significantly decreased overall survival (hazard ratio [HR] 1.91, CI 1.29-2.18, p=0.001 versus HR 2.57, CI 1.62-4.08, p&lt;0.0001). Conclusions: These results provide for the first time evidence for an important role of nuclear NFATc1 overexpression in the molecular pathogenesis of FLT3-ITD positive AML and the induction of drug resistance. These data also underscore that the mutational landscape in AML insufficiently describes AML biology. Disclosures No relevant conflicts of interest to declare.

Current Management of Childhood Acute Myeloid Leukemia.

The outcome for children with acute myeloid leukemia (AML) has improved significantly over the past 30years, with complete remission and overall survival rates exceeding 90 and 60%, respectively, in recent clinical trials. However, these improvements have not been achieved by the introduction of new agents. Instead, intensification of standard chemotherapy, more precise risk classification, improvements in supportive care, and the use of minimal residual disease to monitor response to therapy have all contributed to this success. Nevertheless, novel therapies are needed, as the cure rates for many subtypes of childhood AML remain unacceptably low. Here, we briefly review advances in our understanding of the biology and genetics of AML, the results of recent clinical trials, and current recommendations for the treatment of children with AML.

Research progress of epigenetic regulation and its targeted therapeutics in acute myeloid leukemia

The treatment for acute myeloid leukemia (AML) has not been able to make breakthrough in the last few years due to the limitations of conventional chemotherapy, especially on how to improve the efficacy of recurrent or refractory AML patients, which has become a problem to be solved urgently in clinical.In recent years, with further understanding of molecular biology of AML, some molecular genomic changes with prognostic implications are potential biomarkers for risk stratification and targeted therapy, which caused widespread concern.Researches of the molecular targeted agents of AML are emerging, among them, the epigenetic targeting therapy has made great progress.Epigenetic regulations are reversible which is different from gene changes. Those silent tumor suppressor genes would express again by using appropriate epigenetic agents. The current application of epigenetic drugs mainly includes DNA demethylation agents, histone deacetylase inhibitors and the combination of them. Epigenetics therapies are expected to be treatment options in high-risk AML patient, especially for elderly patients who can′t tolerate intensive chemotherapy.This article reviews literatures on research progress of epigenetic regulation and its targeted therapeutics in AML. Key words: Leukemia, myeloid, acute; Molecular targeted therapy; Epigenomics

Changes in cytogenetics and molecular genetics in acute myeloid leukemia from childhood to adult age groups.

To obtain better insight into the biology of acute myeloid leukemia (AML) in various age groups, this study focused on the genetic changes occurring during a lifetime. This study analyzed the relation between age and genetics from birth to 100 years in 5564 patients with de novo AML diagnosed from 1998 to 2012 (1192 patients from nationwide pediatric studies [AML Berlin-Frankfurt-Münster studies 98 and 2004] and 4372 adults registered with the Munich Leukemia Laboratory). The frequencies of cytogenetic subgroups were age-dependent. Favorable subtypes (t(8;21), inv(16)/t(16;16), and t(15;17)) decreased in general from the pediatric age group (2 to < 18 years; 33%) to the oldest groups (<5% for > 70 years; P < .0001). Unfavorable cytogenetics (-7/del(7), -5/del(5q) or 5p, inv(3)/t(3;3), t(6;9), complex karyotype, 12p, 17p, and 11q23/mixed-lineage leukemia aberrations, excluding t(9;11)) were frequent (42%) in infants (<2 years), had a low frequency in children and young adults (<22%), and increased in frequency up to 36% in patients older than 85 years (P = .01). This was even more significant for complex karyotypes (P ≤ .0001), which also showed a strong increase in the absolute age-specific incidence with age. Interestingly, the frequency of 11q23 abnormalities decreased from infants to older patients. The proportion of clinically relevant molecular aberrations of CCAAT/enhancer binding protein α, nucleophosmin (NPM1), and NPM1/fms-related tyrosine kinase 3-internal tandem duplication increased with age. Altogether, with the exclusion of infants, a significant decrease in the proportion of favorable cytogenetic subtypes and an increase in unfavorable cytogenetics were observed with increasing age. These findings indicate different mechanisms for the pathogenesis of AML; these different mechanisms also suggest directions for etiological research and contribute to the more unfavorable prognosis with increasing age. Cancer 2016;122:3821-3830. © 2016 American Cancer Society.

Suppression of APC/CCdh1 has subtype specific biological effects in acute myeloid leukemia.

The E3 ubiquitin ligase and tumor suppressor APC/CCdh1 is crucial for cell cycle progression, development and differentiation in many cell types. However, little is known about the role of Cdh1 in hematopoiesis. Here we analyzed Cdh1 expression and function in malignant hematopoiesis. We found a significant decrease of Cdh1 in primary acute myeloid leukemia (AML) blasts compared to normal CD34+ cells. Thus, according to its important role in connecting cell cycle exit and differentiation, decreased expression of Cdh1 may be a mechanism contributing to the differentiation block in leukemogenesis. Indeed, knockdown (kd) of Cdh1 in HL-60 cell line (AML with maturation, FAB M2) led to less differentiated cells and a delay in PMA-induced differentiation. Acute promyelocytic leukemia (APL, FAB M3) is an AML subtype which is highly vulnerable to differentiation therapy with all-trans retinoic acid (ATRA). Accordingly, we found that APL is resistant to a Cdh1-kd mediated differentiation block. However, further depletion of Cdh1 in APL significantly reduced viability of leukemia cells upon ATRA-induced differentiation. Thus, low Cdh1 expression may be important in AML biology by contributing to the differentiation block and response to therapy depending on differences in the microenvironment and the additional genetic background.

Abstract A04: Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy

Abstract Metabolic alterations are a cancer hallmark that are typically evaluated in bulk tissues. However most normal tissues and cancers are hierarchically organized and the metabolic requirements of both normal and cancer stem cells are poorly understood, particularly beyond energy metabolism. By analyzing a comprehensive transcriptional roadmap of human hematopoiesis, and comparing this to a leukemia stem cell (LSC) signature developed from 84 human acute myeloid leukemia (AML) samples, we found that: (1) several metabolic pathways, specifically in bioactive lipids, distinguish normal hematopoietic stem cells (HSC) from progenitors; and (2) while LSC are similar to HSC, specific metabolic pathways are more comparable to those of normal progenitors. We defined a lipid stem signature of 24 lipid genes, including sphingolipid genes, whose expression is higher in HSC than progenitors. Interestingly, sphingosine-1-phosphate (S1P) is known to play a role in HSC egress, and ceramide vs S1P levels serve as a rheostat to regulate cell growth and survival. To determine if sphingolipids play a functional role in the primitive hematopoietic compartment, we altered sphingolipid signaling by plating sorted populations of HSC or granulocyte-myeloid progenitors (GMP) in methylcellulose containing myriocin, which inhibits the first step of de novo sphingolipid synthesis, or FTY720, a S1P mimetic. Myriocin decreased GMP colony output but did not affect CFC derived from HSC. By contrast FTY720 affected HSC-derived CFC but not those from GMP, suggesting differential sensitivity to sphingolipid pathway inhibition between stem and progenitor cells. In vitro treatment of lineage depleted cord blood (Lin- CB) with myriocin for 8 days limited only myeloid differentiation compared to control treated cells. In contrast, FTY720 treatment reduced levels of immunophenotypic stem cells, erythroid and myeloid cells, as would be expected with inhibition of S1P proliferative signaling. However, in vitro FTY720-treated Lin- CB cells exhibit 16-week engraftment capacity comparable to that of controlled-treated cells, suggesting that any effects on HSC function are reversible after drug withdrawal. Furthermore, in vivo treatment with FTY720 in mice with established CB grafts did not decrease engraftment. Our lipid stem signature is enriched in LSC gene expression profiles from our 84 AML cohort by GSEA analysis, suggesting that differences in lipid metabolism may also exist in LSC vs. non-LSC. To determine if sphingolipids play a functional role in AML biology, we transplanted mice with peripheral blood cells from 13 AML patients, including those with therapy resistant and relapsed disease, and treated engrafted mice with myriocin or FTY720. We observed heterogeneous responses in our cohort, with reduction of leukemic burden in 3 and 5 samples following treatment with FTY720 and myriocin, respectively. Remarkably, serial transplantation of FTY720 responders into untreated secondary mice at limiting dilution demonstrated decreased LSC frequency in FTY720-treated primary mice compared to vehicle-treated controls, whereas myriocin responders showed no alteration of LSC frequency. These results suggest that FTY720 but not myriocin treatment affects LSC number and/or function. To stratify responders from nonresponders, we performed transcriptional analysis of untreated patient samples and compared these data to transcriptional signatures generated from the normal and AML hierarchy. Bioinformatic analysis demonstrated that FTY720 responders had an enriched LSC signature compared to nonresponders. In contrast, myriocin responders exhibited a strong GMP signature compared to nonresponders. Thus, normal human hematopoietic stem and progenitor cells display a variable dependence on sphingolipid biology that is also distinct between LSC and HSC, pointing to targeting of bioactive sphingolipids as a novel therapeutic strategy in AML to eradicate LSC while sparing HSC. Citation Format: Stephanie Z. Xie, Elisa Laurenti, Robin Ferrari, John E. Dick. Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A04.

BCL11A expression in acute myeloid leukemia

BackgroundBCL11A encodes a C2H2 type zinc-finger protein. During normal haematopoietic cell differentiation BCL11A expression is down-regulated. Data in mice suggest up-regulation of BCL11A is involved in the pathogenesis of myeloid leukaemias. BCL11A expression in persons with acute myeloid leukaemia (AML) is not systematically studied. ObjectiveInterrogate associations between BCL11A expression at diagnosis and clinical and laboratory valuables and outcomes in newly-diagnosed persons with AML. MethodsWe determined BCL11A mRNA levels in bone marrow and blood mononuclear cells in 292 consecutive newly-diagnosed subjects with AML by reverse transcript and real-time polymerase chain reaction. Data were compared to mRNA levels in bone marrow cells of normals. ResultsSubjects with BCL11A transcript levels at diagnosis exceeding the median value of 2.434 (±3.423 SD; 25th–75th inter-quartile range, 1.33–4.29) had higher WBC levels, a greater proportion of bone marrow myeloblasts, were more likely to be FAB M0 subtype, less likely to be FAB M3 subtype, more likely to be in the intermediate cytogenetic risk cohort, less likely to have a complex karyotype and more likely to have DNMT3AR882 and FLT3-ITD mutations than subjects with transcript levels below the median value. In 89 subjects receiving conventional induction chemotherapy the complete remission rate was 54% (95% confidence interval [CI]; 33, 75%) in the lower BCL11A cohort and 65% (45, 85%; P=0.26) in the higher BCL11A cohort. 3 year survival was 33% (2, 65%) in the lower BCL11A cohort and 15% (0, 39%; P=0.35) in the high BCL11A cohort. ConclusionBCL11A transcript levels at diagnosis was significantly associated with several clinical and laboratory variables. There were also non-significant associations with complete remission rate and survival. These data suggest a possible role for BCL11A expression in AML biology.

A Novel Synthetic Lethal Approach Targeting SIRT6 in Acute Myeloid Leukemia

Background: Acute Myeloid Leukemia (AML) is an incurable disease characterized by a highly unstable genome, resulting in large-scale changes at diagnosis, as well as further evolution contributing to disease progression. However, the mechanisms whereby tumor cells adapt to genomic instability are largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage repair (DDR) machinery. SIRT6 is an important regulator of cellular stress response and genomic integrity. Here we investigated the role of this NAD+ -dependent deacetylase in regulating ongoing DNA damage observed in AML patients.Methods: SIRT6 mRNA level was determined by RT-qPCR in AML patients (n=100) diagnosed at the Hematology Department of University of Genoa (Italy), compared with normal bone marrow derived CD34+ cells (n=5). Correlation studies with clinical and molecular characteristics of these patients were also performed. A panel of different AML cell lines and primary cells, both sensitive and resistant to conventional and novel anti-AML therapies, was used in the study. The anti-leukemic effect of DNA-damaging agents (DDAs) including idarubicin, Ara-C and fludarabine was evaluated in presence of SIRT6 depletion/inhibition by CTG assay and Annexin-V/propidium iodide staining. Mechanistic studies were performed with Western-blotting, lentivirus-mediated shRNAs and immunofluorescence assay. Analysis of DNA DSB repair was done using chromosomally integrated reporter constructs, followed by cytometer analysis.Results: AML patients were grouped into lower and higher SIRT6 expressers according to its median mRNA level. Patients with lower expression had a higher incidence of FLT3-ITD (p=0.034, Wilcoxon signed rank test). No significant association was observed with respect to mutations of NPM1, nor with WT1 and BAALC expression. SIRT6 expression correlated also with adverse clinical outcome in term of event free and overall survival (p=0.035 and p=0.025, respectively; unpaired t test). Based on these data, we evaluated SIRT6 role in biology of AML. We found higher SIRT6 protein level in AML cell lines carrying FLT3-ITD mutation (MOLM-14 and MV 4-11) compared to cell lines harboring other mutations (OCI-AML3, THP-1, KG, NB4, HL60, Nomo1 and U937). Targeting SIRT6 by specific shRNAs weakly reduced AML cell survival compared with control-scrambled cells, by impairing DNA repair efficiency. Indeed, a restricted effect of SIRT6 impairment on DNA damage proteins (H2AX, RAD51, 53BP1, RPA32) was measured. We next examined the therapeutic relevance of SIRT6 inhibition in AML by testing effects of its depletion in combination with genotoxic agents. Remarkably, SIRT6 depletion conferred increased sensitivity of AML cells to idarubicin, Ara-C and Fludarabine. Overall, enhancing genotoxic stress while concomitantly blocking DNA double-strand breaks (DSBs) repair response, may represents an innovative strategy to increase chemosensitivity of AML cells. Further mechanistic studies revealed that SIRT6 acts as a genome guardian in leukemia cells by binding DNA damage sites and activating DNA-PKcs and CtIP by deacetylation, which in turn promotes DNA repair.Conclusion: Genomic instability is present in all hematologic malignancies including AML. Strategies aimed to shift the balance towards high DNA damage and reduced DNA repair by SIRT6 inhibition can decrease AML growth and may benefit patients with otherwise unfavorable outcomes. DisclosuresNo relevant conflicts of interest to declare.

The Role of the Slit-Robo Family in Adult Patients with Acute Myeloid Leukemia

Introduction: SLIT-ROBO is newly discovered ligand-receptor family of neuronal guidance molecules. Recently, it has been proved that these proteins are involved in both, physiologic and pathologic angiogenesis. In animal models, it was shown both, pro-and antiangiogenic of SLIT-ROBO signaling. Moreover, the interaction of SLIT ligands with their roundabout receptors (ROBO) results in promotion of apoptosis, adhesion and blocking of cell cycle. There is evidence that SLIT-ROBO proteins are involved in pathogenesis of solid tumors, both in angiogenesis dependent and independent way. The role of SLIT-ROBO proteins in biology of acute myeloid leukemia (AML) remains unknown.The only two hitherto published studies considering ROBO4 expression in AML have revealed its increased expression in the blasts cells.The aim of the study was to evaluate the role of SLIT-ROBO proteins in AML. The expression of SLIT ligands, and their receptors ROBO was assessed in bone marrow of newly diagnosed AML patients and in the control group. The expression level of the proteins was correlated with known prognostic factors, response to treatment and overall survival (OS), as well as angiogenesis activity.To our knowledge, it has been the first study investigating the whole family of SLIT-ROBO proteins in AML.Methods: Expression SLIT-ROBO proteins was assessed in bone marrow biopsy specimens of 79 newly diagnosed AML patients with median age 59 years [18-87]. The paraffin-embedded tissue blocks were retrieved and subjected to immunohistochemistry for SLIT ligands (SLIT1, SLIT2, SLIT3), and their receptors ROBO1, ROBO2, ROBO3, and ROBO4. The positive blasts cells were semi-quantitatively analyzed according to previously published methods (Perrone et al, 2006). For the purpose of analysis the patients were divided into "low-expressers" and "high-expressers". Concurrently, all samples were immunostained for CD34 to calculate microvessel density (MVD) as an equivalent of angiogenesis. The control group was composed of 23 BM biopsies form patients with newly diagnosed lymphoma without bone marrow involvement.Results:Expression of ROBO receptors and SLIT ligands in AML patients and in the control group.In our study higher expression of ROBO1, ROBO2, and ROBO3 was observed more often in AML patients compared to the control group (p<0.0001, p<0.001, and p=0.09, respectively, Fig 1.). In contrast, low expression of SLIT1, SLIT2, and SLIT3 ligands has been shown more often in AML than in control BM samples (p<0.0001, p=0.003, and p=0.001, respectively,Fig.2.). Higher expression of ROBO1, ROBO2, and ROBO3 was more often in AML patients ≥60 years (p=0.04, p=0.008, and p=0.02, respectively).Conversely, low expression of ROBO4 was more often observed in elderly AML (p=0.06). The majority of patients with de novo AML had low expression of SLIT1 and SLIT2 (p=0.053 and p=0.055, respectively). As to ROBO, higher expression of ROBO2 in the group with secondary AML was more frequent (p=0.09). No significant correlations between the SLIT-ROBO proteins' expression,neither cytogenetic risk group nor clinical stage parameters such as WBC, hemoglobin level, proportion of leukemic blasts in BM, or LDH activity were found. Similarly, neither of the SLIT-ROBO proteins influenced the complete remission rate (CR) and overall survival (OS).Relationship between SLIT-ROBO expression and angiogenesis activity in AML patients and control group.Significantly higher MVD in BM of AML patients than in control group (Me 51 [9-140] vs 16 [4-78], p<0.0001) has been observed. ROBO4was the only protein that expression correlated significantly with MVD. Higher expression of ROBO4 was associated with higher MVD in both, AML and the control group (p=0.05 and p=0.01, respectively).Conclusions:SLIT-ROBO family members play a role in biology of AML. ROBO4 is involved in both, physiologic and pathologic angiogenesis A better understanding of SLIT-ROBO signaling pathway in leukemic blasts may create new optionsfor AML therapy.Acknowledgments:AG and DJ-K both equally contributed to the study.This work was supported by grants from Medical University of Lodz, Lodz, Poland (502-03/1-093-01/502-14-077 and 503/1-093-01/503-11-001). [Display omitted] [Display omitted] DisclosuresRobak:Eisai Inc: Research Funding. Wierzbowska:Janssen, Celgene: Consultancy.

Pkc Epsilon Regulates Mitochondrial Redox Biology to Support the Differentiation Blockade in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by the clonal expansion of myeloid progenitors that have impaired differentiation capacity. Determining the molecular machinery that regulate the survival and differentiation blockade of AML cells could serve as a foundation for designing novel therapies. PKCε is a serine-threonine kinase belonging to the subgroup of the Protein Kinase C family called Novel PKCs. Aberrant PKCε expression and activation is associated with the pathogenesis and chemotherapy resistance of many solid cancers. However, the contribution of PKCε in blood malignances such as AML is not well defined.To evaluate the role of PKCe in AML biology, we employed short hairpin RNA (shRNA)-mediated approaches to down-regulate PKCε expression in human and murine AML cell lines. We found that shRNA-mediated knockdown of PKCε significantly reduces the in vitro expansion of several human AML cell lines (MOLM-14, NOMO-1, OCI-AML3, THP-1 and U937). We also observed that blocking PKCε induces caspase-3 cleavage and increases the number of annexin V-positive cells (P<0.05), suggesting that PKCε antagonizes AML cell apoptosis. Additionally, we have also found that prior to cell death, AML cells expressing PKCε-targeting shRNAs display characteristics of myeloid differentiation. Specifically, down-regulation of PKCε results in altered expression of the myeloid differentiation transcription factors C/EBPa and PU.1 and increased expression of the mature myeloid marker CD11b (P<0.001). Moreover, upon PKCε inhibition, AML cells acquire morphological changes associated with differentiation, such as increased cytoplasmic volume, granule formation and nuclear segmentation.Interestingly, we observed similar phenotypic changes when we inhibited PKCε expression in murine AML cell lines driven by the leukemogenic fusion protein MLL-AF9 alone (MLL-AF9) or in combination with the internal tandem duplication mutation of murine Flt3 (MLL-AF9;Flt3-ITD). Specifically, we observed that PKCε down-modulation significantly reduces murine AML cell survival (P<0.001) and colony formation in methylcellulose (P<0.001) of both MLL-AF9 and MLL-AF9;Flt3-ITD cells compared to non-targeting shRNA-expressing cells. We are currently investigating how PKCε inhibition impacts AML progression in vivo using mouse models of AML driven by MLL-AF9 or MLL-AF9;Flt3-ITD.At the molecular level, we have found that PKCε is a key regulator of reactive oxygen species (ROS) biology in AML cells. Specifically, using a fluorogenic probe (CellRox) that indiscriminately detects most types of ROS, we have observed that PKCε knockdown in human AML cell lines results in increased steady-state levels of intracellular ROS compared to shRNA control cells (P<0.002). Total intracellular ROS levels are influenced by the production and clearance of distinct ROS types in various cellular compartments. To further characterize the localization and specific type(s) of ROS regulated by PKCε, we utilized four redox-sensitive GFP (roGFP) probes, which allow for direct measurement of cytoplasmic and mitochrondrial glutathione redox potential (Grx1-roGFP-Cyto and Grx1-roGFP-Mito, respectively) and hydrogen peroxide (H2O2) levels (Orp1-roGFP-Cyto and Orp1-roGFP-Mito, respectively) in live cells. Down-modulation of PKCε in NOMO-1 and OCI-AML-3 cells expressing each of these roGFP constructs resulted in a significant increase in the oxidation of Grx1-roGFP (P<.0007) and Orp1-roGFP-Mito (P<0.02) but not either of cytoplasmic constructs, suggesting that PKCε regulates the production of ROS in the mitochondria of AML cells. Since increased H2O2 production and glutathione oxidation results from increased superoxide (O2-) production in mitochondria, we next evaluated the impact of PKCε on O2- production. Using a fluorogenic probe (MitoSOX) that specifically detects O2- in live cells, we found that PKCε down-modulation increases the production of O2- in AML cells (P<0.05). Our future studies are focused on determining the precise molecular events that connect alterations in redox biology with the survival and differentiation of AML cells.Collectively, these results uncover the previously unrecognized role of PKCε as a critical regulator of mitochondrial redox biology and supporter of cell survival and impaired differentiation in AML. DisclosuresNo relevant conflicts of interest to declare.

Comparison of Age at Diagnosis, Cytogenetic Risk, and Overall Survival Between Acute Myeloid Leukemia Patients of White and South Asian Race/Ethnicity in the United States

Introduction:Acute myeloid leukemia (AML) is more frequent among older patients in the United States (US), with a median age at diagnosis of 67 years old. A recent case series of AML patients from India reported a median age at diagnosis of 40 years old, suggesting that the pathogenesis of AML may differ between these populations (British Journal of Haematology 2015;170:110). In this study, we examined whether differences exist in the age at diagnosis, cytogenetic risk, and overall survival (OS) of White and South Asian patients diagnosed with AML in the US.Methods:We used the 1973-2012 Surveillance, Epidemiology, and End Results Program (SEER) database to identify adults, age 20 years or older, diagnosed with AML between 2000 and 2012. We included patients with documented race/ethnicity and known age at diagnosis. We compared age at diagnosis, cytogenetic risk, and OS according to White or South Asian race/ethnicity, based on patient surname as defined by SEER. We stratified age at diagnosis into age groups, defined as 20-24, 25-34, 35-44, 45-54, 55-64, and >65 years old, to compare the White and South Asian populations. Using the 2012 US Census population age distributions, we directly standardized the distribution of age at diagnosis of AML in SEER, weighted according to the age distribution of the total White and South Asian populations in the US. We categorized SEER-reported cytogenetic profiles as having favorable or adverse prognosis based on accepted definitions. We compared cytogenetic risk and OS between White and South Asian populations according to stratified age group at diagnosis. Differences in age at diagnosis were calculated using the Mann-Whitney test. OS was compared by the Log-rank test and estimated by the method of Kaplan and Meier. P-values <0.05 were considered significant.Results:39,192 patients, age 20 years old and above, were diagnosed with AML from 2000 to 2012 and had documented race/ethnicity at diagnosis in the SEER database. South Asian patients in the US were diagnosed with AML at a significantly younger age compared to White patients (Figure 1A, median age at diagnosis of 57 vs. 69.5 years old for South Asians (n=265) vs. Whites (n=33,419), p=<0.0001). Along with younger age at diagnosis, South Asians had a greater reported frequency of favorable cytogenetic risk (17.7% vs. 9.7% favorable cytogenetic risk for South Asians vs. Whites). Analysis of the demographics of the US population also showed that the South Asian population was significantly younger than the White population (median age of 40 vs. 50 years old for South Asians (n=2,447,009) vs. Whites (n=172,366,410), p=<0.0001). Direct standardization of the age at AML diagnosis with the age distributions of White and South Asian census populations in the US abrogated the differences in age at diagnosis between these groups (Figure 1B, p=0.8718). Standardization by age distribution also narrowed the difference in favorable cytogenetic risk between Whites and South Asians (17.9 vs. 19.1 cases per one million people, respectively). OS was not different between Whites and South Asians in the 20-49 year old age group (median OS: 46 vs. 60 months for Whites (n=5,272) vs. South Asians (n=96), p=0.4986), the 50-64 year old age group (median OS: 13.5 vs. 16 months for Whites (n=6,066) vs. South Asians (n=62), p=0.5088), or the >65 year old age group (median OS: 3 vs. 4.5 months for Whites (n=13,692) vs. South Asians (n=66), p=0.8491).Conclusions:In the US, AML patients of South Asian descent are diagnosed at a younger age and have more favorable cytogenetic risk profiles as compared to their White counterparts, which is of epidemiologic importance. Nevertheless, these findings appear to reflect the younger age distribution of the entire South Asian population as compared to the total White population in the US, rather than a difference in the inherent biology or pathogenesis of AML. These data highlight the importance of directly standardizing age distributions in population outcomes research. [Display omitted] DisclosuresFathi:Agios Pharmaceuticals: Other: Advisory Board participation; Merck: Other: Advisory Board participation; Seattle Genetics: Other: Advisory Board participation, Research Funding.

Histone Deacetylase Inhibitors Induce microRNAs Targeting BTK in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a highly heterogeneous disease primarily affecting the elderly. Despite considerable progress in understanding the biology of AML, the prognosis of the majority of patients still remains poor due to lack of progress in improving therapy over the past 3 decades, thus creating the need to identify new pharmacological strategies.Bruton's Tyrosine Kinase (BTK) has been demonstrated to be functionally important in a variety of hematological malignancies and several groups have recently shown that BTK is highly expressed and constitutively active in AML cell lines and in a subset of AML patients. Moreover, treatment with ibrutinib, a covalent irreversible inhibitor of BTK, can reduce proliferation, blast adhesion to bone marrow stromal cells and migration of both AML cell lines and primary AML blasts. Validation of this target also occurred with shRNA experiments confirming the specificity of BTK for growth inhibition of AML.Dysregulation of microRNA (miRNA) expression contributes to the pathogenesis of numerous human malignancies including AML, where multiple miRNAs have been associated with specific cytogenetic and molecular subsets of the disease.Using a target prediction algorithm we identified several miRNAs as potential regulators of BTK (miR-147b, miR-210-3p, miR-425-5p, miR-1253, miR-4269, miR-4667-3p). We performed 3′UTR luciferase reporter assays in the AML cell line OCI-AML3 and found that relative luciferase activities were significantly decreased (20-30%) in cells transfected with miR-147b, miR-425-5p, miR-4269 and miR-4667-3p mimics. Flow cytometry and western blotting analysis confirmed that BTK protein expression was significantly down-regulated by all the predicted miRs except miR-1253.Previous data from our lab have shown that histone deacetylase (HDAC) 1 and 2 become recruited to multiple microRNA promoters, including the ones targeting BTK. To determine the consequence of HDAC inhibition on BTK signaling, we exposed primary blasts from 14 AML patients to 10nM of the pan-deacetylase inhibitor panobinostat (LBH589) for 48h. Our results showed that panobinostat induced a 2-23 fold increase in the expression miR-147b, miR-210-3p, miR-425-5p, and miR-4667-3p in most of the patients and a time-dependent depletion of total BTK protein and downstream BTK signaling (phospho- and total ERK and phospho- and total AKT) in about half of them. BTK protein at 24h directly correlated with BTK and HDAC1 mRNA expression at 24h and miR-210 induction at 24h inversely correlated with BTK mRNA levels at 24h. Moreover, 2 days of incubation with 10nM panobinostat is able to induce robust cell death in AML primary samples (9.6-92% apoptosis).We hypothesized that HDAC inhibitors would cooperate with kinase inhibitors of BTK to synergistically abolish BTK signaling and induce death in AML cells. Therefore, we treated OCI-AML3 and MV4-11 cell lines with 0.40 µM abexinostat, 1 µM ibrutinib for 2h alone followed by washout, or a combination of 0.40 µM abexinostat with 1 µM ibrutinib for 2h, and viability was measured at 48h. The combination of abexinostat and ibrutinib induced greater than additive cytotoxicity compared to either abexinostat or ibrutinib alone. Exposure to ibrutinib alone for 2h followed by washout inhibited the kinase activity of BTK (measured by the auto-phosphorylation on Y223) but did not affect total BTK protein. However, the combination of abexinostat and ibrutinib showed both loss of BTK kinase and decrease in total BTK protein, in addition to attenuation of BTK signaling, thus confirming the dual targeting of BTK via independent mechanisms.Our results provide the rationale for the clinical evaluation of ibrutininb in combination with histone deacetylase inhibitors in AML patients. DisclosuresNo relevant conflicts of interest to declare.