ALBERT

Beschreibung: Acute myeloid leukemia (AML) is organized in a hierarchy with a rare population known as leukemia stem cells (LSC) capable of self-renewal and propagation of the disease. Characterization of the unique phenotypes and complex signaling pathways in LSCs that survive induction chemotherapy is essential for understanding of the mechanisms of chemoresistance and designing the strategies to eliminate residual leukemia clones. In this study, we compared signaling profiles of distinct phenotypic AML subsets in paired bone marrow (BM) samples collected at diagnosis and after achieving the complete remission (CR). Cell surface characteristics and signaling pathways activated within sub-populations of AML samples were defined using the novel technology of time-of-flight mass cytometry (CyTOF) that has the ability to perform up to 100 mutiparameter assays in single cells (Bendall et al, Science 2011). First, we validated CyTOF measurements by performing cross-comparisons of surface markers and intracellular proteins measured in AML cells with traditional multi-parametric flow cytometry (FCM). Frequencies of CD123+CD99+ population within CD34+CD38- cells were 73.7%±1.8% and 78.5%±3.7% by CyTOF and FCM. Patterns of specific activation of the intracellular proteins pSTAT5, pERK1/2 and pAKT by GM-CSF, PMA and SCF, and inhibition by selective kinase inhibitors showed excellent cross-platform consistency between CyTOF, FCM and immunoblotting. Next, mononuclear cells of 5 paired AML (at diagnosis and in CR) and of 3 normal BM (NBM) were stained with 11 cell surface markers (CD34, CD38, CD123, CD99, CD45, CD33, CD117, CD7, CD4, CD90 and CD133) and 8 intracellular markers (p-4EBP1, p-NF¦ÊB, p-STAT3, p-AKT, p-mTOR, p-ERK, p-S6 and p-STAT5). A SPADE (spanning-tree progression analysis of density-normalized events) (Qiu et al, Nat Biotechnol. 2011) tree plot was generated, representing clustered expression of the cell-surface antigens. Boundaries and annotations of the AML cells were manually defined to represent distinct cell subsets (Figure 1). We used the pooled data from NBM samples, which showed identical patterns, as a reference. SPADE analysis revealed several subsets unique to the diagnostic AML samples, which were eliminated by chemotherapy; and phenotypically distinct subsets in diagnostic samples that persisted in CR. Notably, a subset defined by the “traditional LSC” markers (CD45dimCD34+CD38lowCD90-CD33-CD117+; annotation #2) was readily identified in diagnostic samples and was significantly reduced by induction chemotherapy in 2 of the 5 AML samples. In one of these samples we identified a distinct subset co-expressing LSC markers CD45dimCD34+CD38lowCD33-CD117-CD99lowCD133low (annotation #3) that was present in both diagnostic (1.1%) and CR (1.7%) BM; this subset may have contributed to the MRD detected by standard leukemia-associated immunophenotypes.Figure 1The tree plot was generated using 11 cell surface proteins in AML and NBM, and colored by the median intensity of individual markers (CD34 is shown). Phenotypes of each annotation are indicated.Figure 1. The tree plot was generated using 11 cell surface proteins in AML and NBM, and colored by the median intensity of individual markers (CD34 is shown). Phenotypes of each annotation are indicated. We next investigated intracellular signaling pathways in antigen-defined AML subpopulations using CyTOF. Activation of p-AKT and pS6 showed similar pattern in subsets defined by annotations 1, 9 and 10 at diagnosis (Figure 2A), and was largely reduced in the CR BM. In turn, activation of p-4EBP1 and p-mTOR were observed in multiple subsets (#1-5 and 9-11) in all diagnostic AML samples, especially in a subset 1 characterized by the “Progenitor” phenotype, and remained heightened in the CR samples (Figure 2B).Figure 2The heat map of the average expression of intracellular proteins in selected populations from individual samples. (A) Each column represents individual sample, and each row reflects expression of a certain protein for each annotation. (B) Signaling pathways in annotation #1 in individual samples.Figure 2. The heat map of the average expression of intracellular proteins in selected populations from individual samples. (A) Each column represents individual sample, and each row reflects expression of a certain protein for each annotation. (B) Signaling pathways in annotation #1 in individual samples. In summary, using CyTOF and SPADE, we characterized phenotype-specific intracellular signaling pathways in AML samples at diagnosis and in CR. Persistent activation of p-mTOR and p-4EBP1 are identified in the subpopulations of AML progenitors in CR, and may present the potentially targetable pathways in AML. The study is ongoing with prospective CyTOF analysis of a larger set of paired AML samples at diagnosis, CR and relapse coupled with the molecular analysis of the distinct subpopulations. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Outcomes of acute myeloid leukemia (AML) remain poor and warrant the development of novel therapeutic agents. CD123 (interleukin-3 [IL-3] receptor alpha subunit), is overexpressed on AML blasts and leukemia progenitor/stem cells (LSCs) compared to normal hematopoietic cells (Jordan et al. Leukemia 2000). SL-101 is a novel anti-CD123 antibody-conjugate comprised of anti-CD123 scFv fused to a truncated and optimized pseudomonas exotoxin (PE) lacking its native targeting domain. We have previously demonstrated SL-101's cell killing efficacy in AML cell lines (Han et al. ASH 2013). Here we report the anti-tumor efficacy of SL-101 against primary AML cells and the underlying mechanisms of its cytotoxicity. Fourteen genetically diverse primary AML samples were treated with various doses of SL-101 for 48 h. Most samples express high levels of CD123 (median 89.9%, range 20.4-99.3%) and intermediate levels of CD131 (median 54.0%, range 9.0-91.1%), the IL-3 beta subunit. SL-101 was highly active against AML samples, with an IC50 of 0.19 µg/ml (range 0.003 - 0.98 µg/ml). No significant correlation was found between SL-101 activity and levels of CD123 or CD131 (n=12). SL-101 also selectively and significantly suppressed AML colony formation (69.5% ± 15.0% inhibition of total colonies, n=7) while sparing normal bone marrow (5.6% ± 3.3% inhibition, n=4; p=0.0001) (Fig. 1A). We next investigated the mechanisms of the cytotoxic activity of SL-101. Using annexinV/DAPI flow cytometry, we first evaluated the induction of apoptosis in AML blasts and phenotypically defined CD45dimCD34+CD38-CD123+ LSCs. After 48 h treatment, SL-101 at 1.0 µg/ml induced higher specific apoptosis in the AML LSCs (51.2% ± 25.4%) than in blasts (39.4% ± 19.0%, p=0.006, n=10). Quantification of the annexinV–/DAPI– viable cells using counting beads demonstrated further reduction of cell numbers by SL-101 (LSCs 72%, blasts 64.6%), indicating additional mechanisms of cell growth inhibition. It was recently demonstrated that upon internalization, PE traffics through the endoplasmic reticulum to the cytosol, where it inactivates protein synthesis by catalyzing ADP ribosylation of elongation factor 2 and causes non-apoptotic cell death (Wayne et al. Blood 2014). To examine the contribution of the direct inhibition of protein synthesis by PE, we first studied SL-101 internalization utilizing DyLight 680-labeled SL-101 by flow cytometry and fluorescence imaging. In CD123-expressing AML cell lines MV4-11 and MOLM13, the intracellular median intensity of DyLight 680 signal increased 5.4- and 3.5-fold, respectively, within 4 h of treatment and 22.3- and 16.3-fold after 24 h (Fig 1B). Fluorescence imaging confirmed cytosolic localization of SL-101 in both cell lines, demonstrating efficient cellular uptake of SL-101. We also examined the efficacy of SL-101 in inhibiting nascent protein synthesis in MV4-11 cells using an AHA Alexa Fluor 488 protein synthesis assay. SL-101 significantly reduced protein synthesis (40.3%, p=0.0005) within 4 h (Fig 1C), even at low concentrations (0.01 µg/ml), which was comparable to the positive control cycloheximide (44.8% ± 7.9%, p=0.0001). These findings confirmed the potential of SL-101 to efficiently internalize and promote cell death through protein synthesis blockade. We further investigated the ability of SL-101 to inhibit intracellular signaling in response to IL-3. To this end, cytokine-dependent Mo7e leukemia cells were serum starved and pre-treated with SL-101 at 1.0 µg/ml overnight, followed by stimulation with IL-3. SL-101 significantly suppressed IL-3-induced activation of p-STAT5 (57.1% ± 2.6% inhibition, p=0.003) and modestly inhibited p-AKT (17.4% ± 5.4% inhibition, p=0.04), but not p-ERK signaling (Fig. 1D). In summary, our data demonstrate that the novel anti-CD123 antibody-conjugate, SL-101, is highly active in AML and induces growth arrest and apoptosis in AML blasts and LSCs by inhibiting protein synthesis and interfering with IL-3 signal transduction pathways. Ongoing studies that will be reported at this meeting investigate the in vivo anti-leukemia efficacy of SL-101 in NSG mice engrafted with primary AML cells. In conclusion, SL-101 is a novel, potent antibody-conjugate directed against AML blasts and LSCs, and our studies warrant further development of this agent. Figure 1 Figure 1. Disclosures Rowinsky: Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics: Employment, Equity Ownership.

Beschreibung: BACKGROUND: AML is a group of clinically heterogeneous diseases. We hypothesized that heterogeneous presentation of AML is a reflection of equally heterogeneous genetic process during the leukemogenesis. METHODS: 536 AML patients (pts) bone marrow samples were analyzed by targeted capture exome sequencing of 295 genes. Extensive clinical-genotype correlation was performed using well annotated clinical data. RESULTS: The median age of the cohort was 62 years (IQR: 51-72) including 297 (55%) elderly (age ≥60), and 239 (45%) young (age

Beschreibung: The function of wild-type (wt) p53 in acute myeloid Leukemia (AML) is suppressed by MDM2, MDM4 and XPO-1 (Andreeff et al, Exp Hematol, 2016). We propose that wt p53 protein misfolding and cytosolic localization are contributing to its inactivation in AML. Immunofluorescence staining with OpalR TSA amplification demonstrated that p53 is localized both in the nucleus and in the cytosol of AML cells with prominent para-nuclear accumulation. We show here that misfolded wt p53 is localized mainly in the cytoplasm of AML cells, similar to what we reported for mutant (mt) p53 previously (Zeng et al, Blood, 2016). p53 misfolding promotes its aggregation which was recently reported as a novel mechanism promoting loss of its anti-tumor functions (Xu et al, Nat Chem Biol, 2011; Soragni et al, Cancer Cell, 2016). A pro-aggregating segment in the p53 DNA binding domain is exposed when p53 is misfolded. We showed that ReACp53, a cell permeable peptide designed to inhibit the aggregation of this segment, induced apoptosis in ovarian cancers bearing mt p53 (Soragni et al, Cancer Cell, 2016). We also reported that wt p53 AML cells responded to ReACp53 treatment (Zeng et al, Blood, 2016). ReACp53 eliminated misfolded p53, promoted its mitochondrial translocation and induced rapid apoptosis, suggesting that cytoplasmic misfolded wt p53 is a novel target in AML. MDM2 promotes p53 degradation, and inhibitors of MDM2 such as Nutlin derivatives are currently in trials for AML. These molecules inhibit p53 proteasomal degradation and result in p53-mediated apoptosis, as we demonstrated pre-clinically and in a Phase I trial of RG7112 in AML (Andreeff et al, Clin Cancer Res, 2015). p53 aggregation is initiated by protein misfolding, and progresses with increasing accumulation of misfolded p53. While p53 degradation is promoted by MDM2, binding of MDM2 to p53 causes p53 to misfold (Sasaki et al, J Biol Chem, 2007). This raises concerns about induction of p53 misfolding and consequent aggregation in tumors treated with MDM2 inhibitors, which could diminish therapeutic efficacy. We observed that levels of total and misfolded p53 and protein aggregation as identified by Proteostat positivity were MDM2 inhibitors dose- and time-dependent in wt p53 AML cells. This supports the hypothesis that MDM2 inhibition can cause not only p53 misfolding but also aggregation. Consequently, we show that adding a p53-aggregation inhibitor such as ReACp53 to an MDM2 inhibitor to limit p53 misfolding and aggregation results in increased cytotoxic activity in wt p53 AML. Co-aggregation of mt p53 with p63/p73 proteins carrying similar pro-aggregating segments has been reported (Xu et al, Nat Chem Biol, 2011). Next, we tested whether coaggregation could be an additional factor sequestering and inactivating wt p53. High levels of ΔNp73α, a tumor-promoting isoform of p73, can antagonize p53 function possibly through hetero-tetramer formation (Coutandin et al, Cell Death Differ, 2009), resulting in chemoresistance (Kazushi et al, Subcell Biochem, 2014). We hypothesize that upregulated ΔNp73α could constrain wt p53 through protein co-aggregation causing inactivation. Increased levels of misfolded p53 and protein aggregation were detected in both ΔNp73α-overexpressing HEK293T and MOLM13 (M13) cells. ΔNp73α-overexpressing M13 cells were resistant to MDM2 inhibitor-induced apoptosis compared to controls but sensitive to ReACp53. Treatment with Nutlin-derivatives (RG7388 or DS3032b) did not alter ΔNp73α levels but caused dose- and time-dependent increases in total and misfolded p53 and protein aggregation. HEK293T and M13 cells overexpressing ΔNp73α had higher levels of misfolded and aggregated p53, which we interpret as ΔNp73α providing a "seed" to accelerate p53 co-aggregation due to MDM2 inhibition. This suggests that ΔNp73α-overexpression conferred resistance to MDM2-mediated apoptosis that could be overcome by inhibition of p53 aggregation. Thus, combination of Nutlin derivatives and ReACp53 treatment exerted enhanced cytotoxicity in both cells lines. In conclusion, our data supports cytoplasmic, misfolded wt p53 as a novel target in AML and offers a rationale to combine therapeutic approaches supplementing MDM2 inhibition with p53 aggregation-targeting molecules to increase effectiveness. The model of wt p53 aggregation and coaggregation induced by MDM2 inhibition may apply to other cancer types. Disclosures Andreeff: Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership. Carter:Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Ishizawa:Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed.

Beschreibung: Acute myeloid leukemia (AML) is one of the most aggressive hematological malignancy that originates in the bone marrow (BM). Despite advances in the molecular characterization of AML, factors regulating its progression are still not known. Among several BM niches that support AML growth in the BM, the osteogenic niche has gained attention in recent years owing to its potential role in leukemogenesis. Genetic alterations in osteoprogenitor cells have been shown to induce myeloid leukemia in mouse models. We reported recently that AML cells induce osteogenic differentiation in mesenchymal stromal cells (MSCs) in the BM to facilitate faster AML engraftment in mice (Battula et al., JCI Insight, 2017). However specifics of this osteogenic niche generated by AML are not known. Here we hypothesize that AML expands osteo-progenitor rich niche in the BM, but that the mature bone is reduced. To determine the type of AML-induced osteo-lineage differentiation in the BM, we generated transgenic reporter mice by crossing Osx-CreERt2 mice with Ocn-GFP; ROSA-tdTomato mice. The resulting triple transgenic mice has the genotype of Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. In these mice the tdTomato (red) positive cells represents osteo-lineage cells that originate from Osterix expressing (Osx+) cells, whereas a GFP+ cell represents an osteocalcin-expressing (Ocn+) mature osteoblast. Seven day old triple transgenic mice were injected with tamoxifen to activate Osx-CreERT2 to mark the Osx+ cells with tomato reporter. To investigate the osteogenic cell type that is induced by AML cells in the bone marrow, we implanted murine AML cells with MLL-ENL fusion proteins into Osx-CreERt2;Ocn-GFP;ROSA-tdTomato mice. Three weeks after implantation of AML cells, the femurs and tibia of these mice were dissected and subjected to histological evaluation using fluorescence microscopy. In control BM without AML, the GFP+ (Ocn+) cells were found in the trabecular bone surface as well as the periosteum of the bone, whereas the tdTomato+ (Osx+)cells were found in the marrow and the bone matrix; this suggests that some of the osteocytes originated from tamoxifen-induced Osx+ osteoprogenitor cells. Interestigly, in mice implanted with AML cells, we found a 3-4 fold increase in Osx+ cells in the marrow compared to normal BM (Fig 1A). However, the number of GFP+ cells on the endosteum and trabecular bone surface was reduced, suggesting that AML cells might expand osteoprogenitor cells but not fully differentiated mature osteoblasts. Next, to investigate whether AML cells affect the mature bone, AML PDX cells developed in our laboratory were implanted into NSG mice. The PDX models usually take 12-14 weeks to achieve 〉90% engraftment in the peripheral blood which provides ample time to observe alterations in bone composition. At this stage, the mice were subjected to computed tomography imaging to measure bone architecture, volume (BV), mineral density (BMD) and bone volume fraction (BVF). Interestingly, we observed large bone cavities close to epiphysis and metaphysis areas in the femur and tibia of mice with AML (Fig 1B). In addtion, BMD and BVF in these mice were reduced by 20-30% compared to control mice without leukemia. To validate the bone resorption in these mice, bone histomorphometric analysis was performed on femurs and tibias from mice with and without AML. Masson-Goldner's Trichrome staining revealed a 5- to 10-fold decrease in the trabecular and cortical bone thickness in AML femurs compared to normal femurs. Moreover, measurements of osteoclast activation by tartrate-resistant acidic phosphatase (TRAP) revealed positive staining for osteoclasts on the endosteal surface and massive bone resorption in AML bone compared to normal bone. Mechanistic studies showed that AML cells inhibit osteoprotegerin (OPG) ~10 fold in MSCs, a factor that inhibits the RNAK ligand which in turn activates osteoclasts that breakdown the bone. In conclusion, our data suggest that bone homeostasis is dysregulated in AML by induction of osteogenic and osteolytic activities simultaneously. AML cells induce an osteoprogenitor niche but also activate osteoclasts resulting in osteopenia/osteoporosis in mouse models. In-depth analysis of bone remodeling in AML patients could result in new insights into the pathobiology of the disease and provide therapeutic avenues for AML. Disclosures Andreeff: Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Celgene: Consultancy; Astra Zeneca: Research Funding; Jazz Pharma: Consultancy; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership. Battula:United Therapeutics Inc.: Patents & Royalties, Research Funding.

Beschreibung: Patients with acute myeloid leukemia (AML) continue facing poor long-term survival due to high relapse rate. Persistence of dormant self-renewing leukemia stem/progenitor cells (LSPCs) has been implicated as driver of subsequent relapse, and stem cell gene signatures are associated with poor outcome (Shlush et al. Nature 2017; Ng et al. Nature 2016; Eppert et al Nat Med 2011). Identification of the unique phenotypes and functional proteins in LSCs surviving induction therapy may aid in understanding the mechanisms of chemoresistance and provide novel therapeutic targets in the residual leukemia clones. In this study, we have developed and optimized a comprehensive single-cell mass cytometry (CyTOF) panel, including 36 markers to define LSPCs, with the goal to identify and characterize expression of multiple intracellular signaling pathways and anti-apoptotic proteins in residual AML cells. The validated CyTOF panel was applied in 21 samples collected from 7 AML patients at diagnosis, in remission and at relapse and 5 healthy donors. Data were analyzed using SPADE (Qiu et al, Nat Biotechnol 2011) or Cytofkit (Chen et al. PLoS Comput Biol 2016) tools. We first generated SPADE trees for all diagnostic samples (n=7, Fig 1A), annotating 7 distinct cell populations based on the median expression of selected surface markers as shown in the heatmap (Fig 1B). From these annotations, populations A1, A2 and A6 were positive for CD34 expression, with A6 representing phenotypically the most primitive fraction CD34hiCD38low population (frequency range 0.04%-17.32%). Fractions A1 and A2 expressed more committed myeloid markers, positive for CD135 and CD33 progenitor markers and differentiation markers including CD15, CD11b, and CD7 (Fig 1B). Variability was observed in terms of cell composition, and non-stem fractions A3-A5 were abundant populations in all AML samples except AML2. We further studied the activation/expression of functional proteins in these populations and found that pro-survival BCL-2 protein was highly expressed in the primitive A6 population across AML samples (median intensity 9.0 ± 5.3 in A6 vs 3.5 ± 3.9 in other populations). Variable p-AKT activation was observed in both A6 (4.9 ± 4.2) and differentiated A3-A5 populations (4.5 ± 3.6). We next examined how multi-parametric CyTOF analysis will aid in characterization of MRD populations by comparing samples from 7 patients collected at the time of diagnosis, in remission and at relapse. Using the Cytofkit bioconductor analysis and FlowSOM algorithm, we identified distinct patterns of relapse (Fig 1C). In AML 1-3, major populations were markedly reduced by induction chemotherapy, but residual cells re-grew and contributed to relapse. In AML 4-7, the major populations at diagnosis were eliminated by the therapy, but minor (or undetectable) populations at diagnosis progressed over treatment and represented the bulk of leukemia upon relapse. This finding is consistent with genomic studies that relapse may originate from either the founding clones or subclones that acquire additional mutations (Ding et al. Nature 2012). In AML#3, a major population (cluster 7, CD34−CD38+CD123+CD64+HLA-DR+CD99+, 75.6%) present at diagnosis was identified as persisting in remission at 2.5% by CyTOF analysis but not by conventional MRD flow cytometry and gave rise to the overt leukemia (78.1%) at relapse (Fig 1D). In this patient whose AML harbored mutation in negative MAPK regulator phosphatase PTPN11, persistent AML cells expressed BCL-2, MCL-1, and p-p38MAPK (Fig 1E), consistent with dominant activation of MAPK signaling and anti-apoptotic proteins. We found highly enriched BCL-2 expression and p38MAPK activation in relapse-driving clones in AML5 and AML7, and in diagnostic clone in AML6 (Fig 1F). In summary, using CyTOF, SPADE and Cytofkit analysis tools, we characterized LSPC-specific intracellular signaling pathways in AML samples at diagnosis, in remission and at the time of relapse. Distinct populations were identified to contribute to relapse, indicating that use of additional targeted therapies such as BCL-2 inhibitors may be instrumental post remission to prevent relapse. In conclusion, analysis of the multi-parametric single cell CyTOF mass cytometry may aid in understanding clonal evolution during chemotherapy and identify potential therapeutic targets in individual patients. Disclosures Ravandi: Astellas Pharmaceuticals: Consultancy, Honoraria; Xencor: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; Abbvie: Research Funding; Jazz: Honoraria; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Macrogenix: Honoraria, Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Orsenix: Honoraria; Jazz: Honoraria; Orsenix: Honoraria; Sunesis: Honoraria; Sunesis: Honoraria; Seattle Genetics: Research Funding; Macrogenix: Honoraria, Research Funding. Roboz:Roche/Genentech: Consultancy; Pfizer: Consultancy; Bayer: Consultancy; Celgene Corporation: Consultancy; Astex Pharmaceuticals: Consultancy; Argenx: Consultancy; Roche/Genentech: Consultancy; Janssen Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy; Pfizer: Consultancy; Eisai: Consultancy; Jazz Pharmaceuticals: Consultancy; Orsenix: Consultancy; Astex Pharmaceuticals: Consultancy; Aphivena Therapeutics: Consultancy; Celgene Corporation: Consultancy; Sandoz: Consultancy; Novartis: Consultancy; Bayer: Consultancy; Otsuka: Consultancy; Aphivena Therapeutics: Consultancy; Celltrion: Consultancy; Daiichi Sankyo: Consultancy; Sandoz: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Celltrion: Consultancy; AbbVie: Consultancy; AbbVie: Consultancy; Orsenix: Consultancy; Cellectis: Research Funding; Novartis: Consultancy; Cellectis: Research Funding; Argenx: Consultancy; Eisai: Consultancy; Otsuka: Consultancy. Andreeff:AstraZeneca: Research Funding. Guzman:Cellectis: Research Funding. Konopleva:Stemline Therapeutics: Research Funding.

Beschreibung: Abstract 3469 It is becoming clear that the microenvironment plays a critical role in tumorigenenesis and drug resistance in cancers including Acute Myeloid Leukemia (AML). Mesenchymal stromal cells (MSC) are a key component of the bone marrow (BM) niche where AML cells reside. Our group and others have reported that MSC are critical mediators of leukemic cell engraftment and survival. Much of the emphasis on studies of the role of MSC in the microenvironment has focused on chemokines and cytokine effects especially those involving the SDF-1/CXCR4 axis. While paracrine mechanisms of MSC regulation of leukemic cells are important, in this report we suggest a novel role for MSC in altering AML cells by exosome-mediated transfer of genetic material. It is only within the last 10 years that microvesicles such as exosomes have been implicated in mammalian cell-to-cell communication. In blood biology, this is perhaps best evidenced in the regulation of antigen presenting cells by exosome containing miRs from T cells (Mittelbrunn et al Nat Commun. 2011; 2:282). At present, the role for exosomes in leukemia biology is unknown. Exosomes are derived from late endosomal processing, are enriched in ceramides, and contain proteins that are critical in membrane trafficking such as the tetraspanin CD63. To determine if MSC could transfer exosomes to AML cells, we introduced GFP-tagged CD63 into MSC. Live confocal imaging of MSC revealed that MSC can indeed secrete exosomes. The secreted exosomes are often observed in clusters, likely due to the high ceramide content of the particles. GFP-CD63 containing exosomes from MSC were purified using the Exoquick kit from System Biosciences (Mountain View, CA) and introduced to OCI-AML3 and THP-1 cells. Confocal imaging revealed exosomal uptake by only few leukemia cells; however, cells that contained the microvesicles appeared to contain clusters of these particles. Co-culture of GFP-CD63+ containing MSC with AML derived OCI-AML3 cells or KG-1 cells for 48 hours resulted in transfer of exosomes to leukemic cells as determined by analytical flow cytometry. Roughly 1.5 % of OCI-AML3 and 0.8 % of KG1 cells were found to have incorporated GFP-CD63. Though exosome transfer from MSC to AML cells appears to occur at low frequency, at least under the conditions employed here, the possibility that even a small number of leukemic cells could be altered by this mechanism could have significant ramifications. Introduction of miRs or other non-coding RNAs could profoundly modify the leukemic cells. If the primary site of residence of leukemic stem cells (LSC) is in the BM niche, then MSC could alter LSC thus creating a heterogeneous population of leukemic cells. The question arises how the BM microenvironment of a healthy individual might differ from that of an AML patient. We conducted miR profiling on MSC from normal healthy donors (N = 15) and AML derived MSC (N = 28). Eighteen miRs showed significant differences in expression between the two groups: AML derived MSC were found to express 〉 2 fold more miR-450b-5p, mir-382, and miR-539. On the other hand, healthy donor MSC express 〉 2 fold higher levels of mir-19a, miR-93, and miR-542-5p compared to MSC from AML patients. While it is not known how the exosomes of AML derived MSC differ from those of healthy donors, the difference in miR profiles between the two groups suggests that the miR content of their exosomes could vary and that exosomes from each group could have different effects. Determination of the miR content in the exosomes from each group will shed light on possible mechanisms and analysis is underway. Finally, it is also possible that MSC could use exosomes to alter the microenvironment itself. Preliminary data obtained by confocal microscopy revealed transfer of CD63+ exosomes from GFP-CD63+ MSC to MSC expressing red fluorescent protein (RFP) during co-culture conditions. At this point, the mechanism of transfer is not clear as it is possible that transfer occurs via cell-to cell contact between MSCs and/or incorporation of secreted exosomes by recipient MSC. Still, this data suggests that MSC can exchange genetic materials and that this novel mechanism could act as a plausible means of modifying the microenvironment. In conclusion, exosomal transfer represents a new mechanism in cell-to-cell communication and is active in the leukemia microenvironment with transfer from normal MSC to AML cells or other component cells of the leukemic bone marrow niche. Disclosures: No relevant conflicts of interest to declare.