Primary Pediatric Acute Myeloid Leukemia Research Articles

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML

BackgroundHematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due...

Rational Combinatorial Targeting By Adapter CAR T Cells (AdCAR-T) Prevents Antigen Escape in Acute Myeloid Leukemia (AML)

Targeting AML by chimeric antigen receptor T cells (CAR-T) has been challenging due to promiscuous expression of AML-associated antigens on healthy hematopoietic stem and progenitor cells as well as a high degree of inter- and intratumoral heterogeneity. Consequently, we did not yet see the dramatic clinical successes as in B-phenotypic malignancies treated with CD19 or BCMA directed CAR-T. In the present study, we analyzed inter- and intratumoral heterogeneity of AML-associated antigens in 30 primary pediatric AML samples at single cell resolution. We identified CD33, CD38, CD371, IL1RAP and CD123 to be most frequently expressed. Notably, high variability was not only observed across the different patient samples, but also among leukemic cells of the same patient, which indicates that multiplexed targeting approaches will be required to target all leukemic cells. To address this need, we utilized our modular Adapter CAR (AdCAR) platform enabling precise qualitative and quantitative control over CAR-T function. We show highly efficient and target specific activity for newly generated adapter molecules (AMs) against CD33, CD38, CD123, CD135 and CD371, both in vitro and in vivo. Importantly, we prove that inherent intratumoral heterogeneity in antigen expression translates into antigen escape and therapy failure to mono-targeted CAR-T therapy. For the first time, we demonstrate in a PDX model that rational combinatorial targeting by AdCAR-T can cure heterogenic disease. In conclusion, we elucidate the clinical relevance of heterogeneity in antigen expression in pediatric AML and present a novel concept for precision immunotherapy by combinatorial targeting, utilizing the AdCAR platform.

TRIM13 Is a Novel Cell Cycle Hub Regulated By CHAF1B to Promote Acute Myeloid Leukemogenesis

CHAF1B (Chromatin Assembly Factor 1B) functions as a histone chaperone within the DNA replication machinery. It is almost universally overexpressed in cancers, and we have previously shown that this upregulation is necessary to enforce the differentiation block found in acute myeloid leukemia (AML) through CHAF1B's chromatin binding function. In this study, we found that CHAF1B creates a pro-leukemic state in hematopoietic cells by repressing the expression of the E3 ubiquitin ligase TRIM13. Our data suggest that TRIM13 is a strong promoter of entry into the cell cycle functioning through novel nuclear localization and activating ubiquitination of key cell cycle genes. We propose that repression of TRIM13 by CHAF1B is a critical event for leukemogenesis because unchecked entry into cell cycle leads to loss of self-renewal and eventual exhaustion of the tumor. First, we measured the expression levels of CHAF1B and TRIM13 in several bone marrow aspirates from primary pediatric AML patients using immunohistochemistry. TRIM13 was expressed in healthy marrow and almost undetectable upon leukemic progression. CHAF1B, on the other hand, had low to moderate expression in healthy tissues and substantially higher expression upon leukemic progression and relapse. We found restoration of healthy CHAF1B and TRIM13 expression levels upon resolution of the tumor in matched patient aspirates upon remission. To determine if CHAF1B is capable of directly repressing TRIM13 expression, first we observed CHAF1B occupancy at the TRIM13 promoter by ChIPseq in multiple AML cell lines. Then we found loss of CHAF1B by shRNA resulted in increased TRIM13 transcription, and overexpression of CHAF1B by dox-inducible lentivirus drove TRIM13 transcriptional repression. Overexpression of a CHAF1B chromatin binding deficient mutant (CHAF1BRRAA) was unable to repress TRIM13, suggesting that CHAF1B chromatin binding is necessary for TRIM13 repression. This led us to hypothesize that TRIM13 may be a novel tumor suppressor in AML. To test this hypothesis, we overexpressed TRIM13 in AML cell lines and several patient-derived xenografts (PDXs) spanning many subtypes of AML and observed consistent repression of colony formation, increase in apoptosis, and reduction of leukemic progression in vivo. Conversely, loss of TRIM13 expression by shRNA led to an increase in colony formation in vitro and enhanced leukemogenicity of PDXs and AML cell lines in vivo. To better understand the mechanistic contribution of TRIM13 to myeloid leukemogenesis, we started by identifying potential catalytic targets of TRIM13. Since TRIM13 is a RING-type E3 ubiquitin ligase, we used CRISPR to generate in-frame RING domain deleted alleles of TRIM13 (T13RINGdel) and performed ubiquitin proteomics comparing our T13RINGdel and TRIM13 WT clones. We expected that loss of the catalytic function would lead to stabilization of pro-leukemic proteins as has been shown in solid tumor models. We did not find that, but we did observe a profound reduction in ubiquitination of proteins associated with the cell cycle, including CCNA1, CCNA2, CCNB2, and CDK1. Additionally, the total levels of these cell cycle-promoting proteins was reduced in T13RINGdel cells suggesting the ubiquitination event leads to their stability and activity. We confirmed that our T13RINGdel clones of AML cell lines had reduced entry and progression through cell cycle. Using imaging cytometry, we found a novel nuclear localization of TRIM13 and an expression pattern characterized by increased TRIM13 expression as cell cycle progressed. We confirmed the stabilization/activation relationship between TRIM13 and CCNA1 by restoring CCNA1 expression and confirming loss of the colony formation benefit in T13RINGdel AML cell lines. This study is the first to link CHAF1B, a cell cycle gene, with transcriptional repression of genes that promote cell cycle progression. We believe this relationship is critical for AML cells to support high levels of CHAF1B expression, thereby promoting an immature leukemic state throughout replication, without premature entry into multiple damaging rounds of proliferation.

P387: UNRAVELING LEUKEMIC STEM CELLS MITOCHONDRIAL DEPENDENCY IN PEDIATRIC ACUTE MYELOID LEUKEMIA

Background: Relapse still represents an unsolved problem for children with acute myeloid leukemia (AML), therefore more effective therapeutic strategies are needed for a complete blasts eradication. Leukemic stem cells (LSCs) have been proposed as the therapy-resistant reservoir of cells responsible for failure of antiproliferative chemotherapy and disease recurrence in AML due to their self-renewal capacity and quiescence allowing evasion from chemotherapy. Thus, the identification of LSCs-specific vulnerabilities is needed for more efficacious treatments. Aims: We aim to dissect novel LSCs characteristics to unravel mechanisms of chemoresistance and disease recurrence, and characterize biological properties of LSCs that can be targeted to completely eradicate AML. Methods: To study LSCs derived from primary pediatric AML samples, we based on reactive oxygen species (ROS) content since it was shown that functionally defined LSCs have relatively low levels of ROS. We isolated the lowest 20% of cells fluorescing with CellROX probe (ROS low) and the highest 20% (ROS high) by cell sorting, and analyzed cell cycle, engraftment in mice and RNA sequencing to identify deregulated pathways. Results: After isolating ROS low and ROS high cells, we first assessed classical stemness features. By colony-forming assay, we observed that ROS low and ROS high cells have similar colony-forming capacity, but only ROS low cells preserve this capacity after a second replating whereas the ROS high population does not (p=0.002). By BrdU assay, we found that ROS low cells are relatively dormant, being more in G0/G1 cell cycle phase (41.9%) than the ROS high counterpart (7.9%, p=0.001), also confirmed by CFSE staining, revealing that ROS low cells proliferate more slowly (at day 4, 31% undivided cells in ROS low vs 8% in ROS high). According to our previously published data, we confirmed that ROS low cells express higher levels of CDK6-AS1 (RQ=1.38) and CDK6 genes (RQ=2.69; RQ=1 for ROS high) and that, being more quiescent, are characterized by lower levels of mitochondrial membrane potential by TMRE and lower content of mitochondrial ROS, byproducts of mitochondrial activity, by MitoSOX staining. We also verified that ROS low cells have a lower mitochondrial mass by MitoTracker and TOMM20 staining (mitochondria to nucleus ratio 1.18 vs 1.35 for ROS high, p=0.0005), suggesting that mitochondrial mass and functions might be involved in quiescent cells properties. However, engraftment experiments did not reveal different capacity of ROS low or ROS high cells in recreating leukemia in NSG mice, neither in terms of human CD45+ cells in mice peripheral blood (23.3% vs 41.9% respectively, p=0.12) nor bone marrow infiltration (73% vs 84.8%, p=0.44) at 8 weeks post-engraftment, opening for further dissection of the presence of LSCs within ROS high cells. By RNA sequencing, we found that the top 150 differentially expressed genes between ROS low and ROS high cells are involved in focal adhesion (logP=-13.51), regulation of hemopoiesis (logP=-12.31), cell response to stress (logP=-8.73) and cytokine production (logP=-8.67). Further analyses will confirm the role of these pathways in maintaining ROS low LSCs. Moreover, we identified 5 differentially expressed surface antigens that are under investigation as putative ROS low LSCs markers. Summary/Conclusion: In conclusion, the ROS low strategy permits to identify a quiescent AML subpopulation with peculiar mitochondrial features and transcriptome allowing to pinpoint novel molecular pathways that might be targeted to enhance LSCs clearance.

Small Molecule Screening of Primary Human Acute Myeloid Leukemia Using Co-culture and Multiplexed FACS Analysis.

Ex vivo culture of primary acute myeloid leukemia (AML) cells is notoriously difficult due to spontaneous differentiation and cell death, which hinders mechanistic and translational studies. To overcome this bottleneck, we have implemented a co-culture system, where the OP9-M2 stromal cells support the growth, but most notably limit the differentiation of primary AML cells, thus allowing for mechanistic studies in vitro. Additionally, the co-culture on OP9-M2 stromal is superior in preserving surface marker expression of primary (adult and pediatric) AML cells in comparison to stroma-free culture. Thus, by combining the co-culture with multicolor, high-throughput FACS, we can evaluate the effect of hundreds of small molecules on multi-parametric processes including: cell survival, stemness (leukemic stem cells), and myeloid differentiation on the primary AML cells at a single-cell level. This method streamlines the identification of potential therapeutic agents, but also facilitates combinatorial screening aiming, for instance, at dissecting the regulatory pathways in a patient-specific manner. Graphic abstract: Schematic representation of the ex vivo small molecule screening of primary human acute myeloid leukemia. Irradiated, sub-confluent OP9-M2 stromal cells are plated in half-area 96 wells plates 4-16 h prior to adding primary AML cells. Compounds are added 36-48 h later and effects on cell number, leukemic stem cell population, and myeloid differentiation are quantifed by FACS after 4 days of treatment.

CircRNF220, not its linear cognate gene RNF220, regulates cell growth and is associated with relapse in pediatric acute myeloid leukemia

BackgroundCircular RNAs (circRNAs) constitute a family of transcripts with unique structures and have been confirmed to be critical in tumorigenesis and to be potential biomarkers or therapeutic targets. However, only a few circRNAs have been functionally characterized in pediatric acute myeloid leukemia (AML).MethodsHere, we investigated the expression pattern of circRNAs in pediatric AML using a circRNA microarray. The characteristics, potential diagnostic value, and prognostic significance of circRNF220 were evaluated. A series of functional experiments were performed to investigate the role of circRNF220 in primary pediatric AML cells. Then we investigated the aberrant transcriptional networks regulated by circRNF220 in primary AML cells by RNA-seq. Furthermore, biotin RNA pulldown assays were implemented to verify the relationship between circRNF220 and miR-30a.ResultsWe identified a circRNA, circRNF220, which was specifically abundant in and accumulated in the peripheral blood and bone marrow of pediatric patients with AML. It could distinguish AML from ALL and other hematological malignancies with high sensitivity and specificity. Significantly, circRNF220 expression independently predicted prognosis, while high expression of circRNF220 was an unfavorable prognostic marker for relapse. Furthermore, we characterized the function of circRNF220 and found that circRNF220 knockdown specifically inhibited proliferation and promoted apoptosis in AML cell lines and primary cells. Mechanistically, circRNF220 may act as an endogenous sponge of miR-30a to sequester miR-30a and inhibit its activity, which increases the expression of its targets MYSM1 and IER2 and implicated in AML relapse.ConclusionsCollectively, these findings demonstrated that circRNF220 could be highly efficient and specific for the accurate diagnosis of pediatric AML, with implications for relapse prediction.

Atovaquone Demonstrates Single Agent Activity and an Additive Effect in Combination with Cytotoxic Chemotherapy (Cytarabine and Daunorubicin) in AML

Background: To improve outcomes for pediatric acute myeloid leukemia (AML), identifying and targeting mechanisms of therapy resistance is critical. Recently, the antimicrobial drug atovaquone (AQ) was shown to reduce IL-6/STAT3 signaling in vitro and to improve relapse-free survival in adult AML patients who used AQ for prophylaxis after bone marrow transplant (Xiang et al, Blood, 2016). Clinically, this FDA approved drug is well tolerated, even in patients with organ dysfunction. It has established pediatric dosing, and plasma concentrations at standard doses are typically 40-80µM. We performed in vitro and in vivo experiments to evaluate the efficacy of AQ as a single agent and in combination with standard induction chemotherapy (cytarabine, daunorubicin, and etoposide). In order to ensure clinical relevance, all experiments examined cytotoxicity at clinically achievable doses.Methods: AML cell lines were incubated with vehicle control or increasing doses of AQ (1µM - 30µM) for 24, 48, 72, or 96 hours, and then apoptosis was quantified by Annexin-V staining. Primary pediatric AML cells were exposed to 30µM AQ for the same time periods. To reduce spontaneous apoptosis of primary AML cells over prolonged culture, samples were cultured in HS5 stromal cell-conditioned media (CM) or co-cultured with HS5 stromal cells. For in vivo studies, female NSG mice were injected with 5x105 THP-1 cells expressing firefly luciferase (THP-1.ffluc). Mice were randomly assigned to receive AQ 200 mg/kg daily (n=6) or vehicle (n=5) by oral gavage, starting on day 1, and disease burden was monitored by bioluminescent imaging 3 times a week. A cohort of male mice receiving VC or AQ starting the day of injection with THP-1.ffluc cells were followed for survival (n=7 for each group). For in vitro drug combination cytotoxicity assays, 103 THP-1.ffluc cells per well were plated in 96 well plates with media only, single drugs at increasing concentrations, and AQ+chemotherapy combinations (AQ 1-15uM, AraC 0.1-3uM, Dauno 0.03-0.6uM, VP-16 0.3-3uM). After 48 hours, viability was determined by luminescence. Background luminescence of media-only wells was subtracted from all values and means of 4 replicates were used for analysis. All values were normalized to the cells + vehicle wells, which were set to 0 to quantify the percent affected. The Calcusyn method was used to calculate the combination index (CI). An AQ+chemotherapy combination was considered synergistic if CI<0.8, additive if CI>0.8 and <1.2, or antagonistic if CI>1.2.Results: All 7 AML cell lines demonstrated time and dose-dependent induction of apoptosis in response to 1-30µM doses of AQ. IC50s were within the range of typical clinically achievable doses. For AML cell lines, the IC50s (in µM) after 72 hour incubation were: MOLM13 13.7, MV4-11 14.0, THP-1 15.7, NB4 16.9, Kasumi 1 18.6, HL60 33.7, KG1 98.9. AQ was generally less potent against the CML cell line K562 and the precursor B ALL cell line RS4 (72 hour IC50s were 116.6 and 81.4 respectively). All tested primary samples demonstrated AQ induced apoptosis that increased over time. After subtracting spontaneous apoptosis at each timepoint, the apoptosis induced by AQ at 72 hours for 5 patient samples supported by co-culture were 36, 37, 39, 67 and 87%. Annexin-V+% for the same patient samples supported by CM were 19, 13, 51, 47, and 24%. Bioluminescent imaging of female NSG mice xenografted with THP-1.ffluc cells and treated with single agent AQ daily starting on the day of cell injection demonstrated decreased disease burden compared to control mice (for example on day 21, the average radiance of VC treated mice vs AQ treated mice was 2 x 105 vs 6.3 x 104 p/s/cm²/sr; p=.0048 by ANOVA). Single agent AQ prolonged survival of male xenografted mice with all VC mice succumbing to disease at a median of 63 days vs 1/7 AQ treated mice by day 70 (p=.003 by log-rank test). Our drug combination assays found an antagonistic effect in combination with etoposide, and an additive effect with cytarabine and daunorubicin.Conclusions: Atovaquone induced dramatic levels of apoptosis of AML cells at clinically achievable doses in vitro ; reduced disease burden and improved survival in AML xenografts; and had an additive effect with standard chemotherapy drugs for pediatric AML. Our data support further evaluation of atovaquone for pediatric AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells.

Type 1 regulatory (Tr1) T cells induced by enforced expression of interleukin-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft-versus-host disease while mediating graft-versus-leukemia effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1- RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. In order to examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML.

Clinical Grade Production of Wilms' Tumor-1 Loaded Cord Blood-Derived Dendritic Cells to Prevent Relapse in Pediatric AML After Cord Blood Transplantation.

Hematopoietic cell transplantation (HCT) is a last resort, potentially curative treatment option for pediatric patients with refractory acute myeloid leukemia (AML). Cord blood transplantation (CBT) results in less relapses and less graft-versus-host disease when compared to other sources. Nevertheless, still more than half of the children die from relapses. We therefore designed a strategy to prevent relapses by inducing anti-AML immunity after CBT, using a CB-derived dendritic cell (CBDC) vaccine generated from CD34+ CB cells from the same graft. We here describe the optimization and validation of good manufacturing practice (GMP)-grade production of the CBDC vaccine. We show the feasibility of expanding low amounts of CD34+ cells in a closed bag system to sufficient DCs per patient for at least three rounds of vaccinations. The CBDCs showed upregulated costimulatory molecules after maturation and showed enhanced CCR7-dependent migration toward CCL19 in a trans-well migrations assay. CBDCs expressed Wilms’ tumor 1 (WT1) protein after electroporation with WT1-mRNA, but were not as potent as CBDCs loaded with synthetic long peptides (peptivator). The WT1-peptivator loaded CBDCs were able to stimulate T-cells both in a mixed lymphocyte reaction as well as in an antigen-specific (autologous) setting. The autologous stimulated T-cells lysed not only the WT1+ cell line, but most importantly, also primary pediatric AML cells. Altogether, we provide a GMP-protocol of a highly mature CBDC vaccine, loaded with WT1 peptivator and able to stimulate autologous T-cells in an antigen-specific manner. Finally, these T-cells lysed primary pediatric AML demonstrating the competence of the CBDC vaccine strategy.

Inhibition of Methyltransferase DOT1L Sensitizes to Sorafenib Treatment AML Cells Irrespective of MLL-Rearrangements: A Novel Therapeutic Strategy for Pediatric AML.

Pediatric acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis for which there are few effective targeted approaches, despite the numerous genetic alterations, including MLL gene rearrangements (MLL-r). The histone methyltransferase DOT1L is involved in supporting the proliferation of MLL-r cells, for which a target inhibitor, Pinometostat, has been evaluated in a clinical trial recruiting pediatric MLL-r leukemic patients. However, modest clinical effects have been observed. Recent studies have reported that additional leukemia subtypes lacking MLL-r are sensitive to DOT1L inhibition. Here, we report that targeting DOT1L with Pinometostat sensitizes pediatric AML cells to further treatment with the multi-kinase inhibitor Sorafenib, irrespectively of MLL-r. DOT1L pharmacologic inhibition induces AML cell differentiation and modulates the expression of genes with relevant roles in cancer development. Such modifications in the transcriptional program increase the apoptosis and growth suppression of both AML cell lines and primary pediatric AML cells with diverse genotypes. Through ChIP-seq analysis, we identified the genes regulated by DOT1L irrespective of MLL-r, including the Sorafenib target BRAF, providing mechanistic insights into the drug combination activity. Our results highlight a novel therapeutic strategy for pediatric AML patients.

Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation

AbstractAtovaquone, a US Food and Drug Administration–approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.

In Vivo Evaluation of Mesothelin As a Therapeutic Target in Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) continues to have a cure rate of only 50% despite the use of highly intensive cytotoxic chemotherapy. Transcriptome sequencing of several AML samples by the NCI/COG TARGET AML Initiative identified mesothelin (MSLN) to be highly overexpressed in about one-third of pediatric AML (Tarlock et al., Blood, 128:2873, 2016). Because MSLN is not expressed in normal bone marrow samples (Fan et al., Blood, 130:3792, 2017) and only to a low level in other human organs and tissues, MSLN is an attractive therapeutic target for pediatric AML (Kaeding et al., Blood, 130:2641, 2017). The anti-MSLN antibody-drug conjugate (ADC) anetumab ravtansine (BAY 94-9343) generated by conjugating MSLN-antibody with tubulin inhibitor DM4 (Meso-ADC), and isotype control antibody conjugated with the same drug (Iso-ADC) were used to evaluate the efficacy of MSLN targeting in vivo. MSLN-overexpressing K562 (K562-MSLN) CML cells and MV4;11 (MV4;11-MSLN) AML cells were generated by lentiviral transduction of MSLN cDNA. Cell line-derived xenografts (CDX) were created by injecting the MSLN-transduced or parental (MSLN-) cells into NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when the median percentage of human cells in mouse peripheral blood was greater than 0.5%. K562-MSLN CDX mice treated with Meso-ADC (5 mg/Kg Q3dx3, i.v.) survived a median of 46 days longer than those treated with Iso-ADC (P=0.0011) and significantly longer than comparison groups, including K562-MSLN CDX mice treated with daunorubicin and Ara-C (DA, P=0.0008) or untreated (P=0.0018) (Fig. 1A). Median survival of K562 CDX mice treated with Meso-ADC, Iso-ADC, or untreated was similar (Fig. 1B). MV4;11-MSLN CDX mice treated with Meso-ADC exhibited complete remission and remained disease-free at 1 year post cell injection, with AML cell burden remaining &amp;lt;0.1% throughout the study period (Fig. 1C). In contrast, MV;11-MSLN CDX mice treated with Iso-ADC or untreated succumbed to disease at 72 and 38 days, respectively. Taken together, these results indicate that Meso-ADC was efficacious in reducing leukemia burden, and this effect required MSLN expression in target cells. We have generated a panel of patient-derived xenograft (PDX) lines by transplanting and serially propagating primary pediatric AML samples into NSG-SGM3 mice. The efficacy of Meso-ADC was also evaluated in a systemic PDX model using a MSLN+ PDX line (NTPL-146). NTPL-146 PDX mice treated with Meso-ADC (5 mg/Kg, Q3dx3 -x2 cycles) survived a median of 50 days longer than those treated with Iso-ADC (P=0.0018, Fig. 1D, arrows indicate time when each treatment cycle was initiated). In an independent experiment with NTPL-146 PDX mice, a survival benefit of Meso-ADC treatment over no treatment was observed after 1 cycle of Meso-ADC treatment (5 mg/Kg, Q3dx3, P=0.0019, Fig. 1E). Additionally, a combination therapy strategy with daunorubicin and Ara-C followed by Meso-ADC (DA -&amp;gt; Meso-ADC) resulted in improved median survival over Meso-ADC (P=0.0027) or DA treatment alone (P=0.0018) (Fig. 1E). The disseminated MSLN+ leukemia mouse models described herein support MSLN-targeted antibody-drug conjugate as a potential treatment strategy in MSLN+ AML. Furthermore, we provide the first in vivo demonstration of synergy between MSLN-targeted therapy and conventional chemotherapy in MSLN+ AML, warranting additional investigation to validate and optimize novel strategies for combination therapy. Figure 1 Disclosures Kaeding: Celgene: Employment. Schatz:Bayer AG: Employment. Sommer:Bayer AG: Employment, Equity Ownership.

Functional miRNA Expression Library Screen Identifies miRNAs That Alter Proliferation and Differentiation in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by increased self-renewal of leukemia stem/progenitor cells and failure of differentiation to mature myeloid cells. MicroRNAs (miRNAs, miRs) are short non-coding RNAs whose power stems from the ability to regulate hundreds of targets by interfering with gene expression by degrading messenger RNA or blocking protein translation. It has become evident that miRNAs are important regulators of normal cell processes that are altered in cancer, and restoring or inhibiting miRNAs may be a therapeutic strategy in cancer. MiRNAs play a significant role in normal hematopoiesis and differentiation; however, our understanding of how miRNAs contribute to impaired differentiation and proliferation in acute leukemia remains limited.We have used a combined approach to prioritize miRNAs for investigation for functional roles in leukemogenesis. To identify miRNAs that may play a role in altered cell proliferation or differentiation of AML cells, we used a functional miRNA library screen. We transduced a high-throughput lentiviral library containing 613 precursor miRNAs (Systems Biology) into the AML cell line THP-1 harboring a MLL-AF9 translocation. These cells were then treated with 12-O-tetra-decanoylphorbol-13-acetate (TPA), an agent that induces monocytic differentiation and cell adhesion. After recovery of proliferating cells we compared miRNA expression before and after TPA induced differentiation using quantitative PCR to identify enriched miRNAs (may block differentiation) and miRNAs that drop out (may promote differentiation). To prioritize miRNAs with phenotypic effects that may be relevant to AML, we focused on miRNAs that are differentially expressed in various AML subtypes, using both miRNA-sequencing data from primary pediatric patient AML samples (N=117) and mature miRNA expression of primary adult patient AML samples (N=20).Selected miRNAs were cloned into a lentiviral expression vector and retested in THP-1 cells to assess whether proliferation or differentiation was altered by expression. In line with previously published data we found enrichment of miR-9-1 and members of the miR-17-92 cluster, and its paralogs miR-106a-363 and miR-106b-25, as inhibitors of myeloid differentiation. In addition, we have validated three miRNAs (miR-590, miR-550A1 and miR-550A2) with previously uncharacterized roles that block differentiation and increase proliferation of THP-1 cells. These phenotypes were confirmed in other AML cell lines, including NB4 cells (PML-RARA translocation) and U937 cells (MLL-AF9 translocation). Increased expression of either miR-590 or miR-550A2 enhances MAPK signaling, as detected by increased phospho-ERK1/2, which influences cell proliferation. We have identified that PPP2R2A, the regulatory B55 alpha subunit of the tumor suppressor protein phosphatase 2A (PP2A), is a miR-590 target. Regulation of this complex by miR-590 could contribute to alterations in MAPK/ERK signaling. To identify additional miR-590 and miR-550A putative targets, we profiled gene expression changes by microarray (Illumina Human HT-12v.4) in THP-1 and NB4 cells expressing miR-590 or miR-550A2 versus control cells. Validation of identified putative targets, as well as functional investigations assessing the impact of the identified miRNAs and targets on AML cell proliferation or differentiation, are underway.In conclusion, functional miRNA screening is an important tool to prioritize differentially expressed miRNAs identified in primary AML patient samples that alter differentiation and proliferation. Using this approach we have identified new roles for miR-590 and miR-550A2 in AML. Our goal is to determine mechanistically how these miRNAs block differentiation in AML cells and to establish how to target these pathways therapeutically to promote differentiation and AML cell death. DisclosuresNo relevant conflicts of interest to declare.

P53 Suppression Mediates Blasts Survival, Proliferation and CD34 Maintenance in Pediatric Acute Myeloid Leukemia

Aberrations in the DNA damage responses contributes to the uncontrolled proliferation and therapy resistance in many cancers. Acute myeloid leukemia (AML) presents enhanced DNA damage as compared to MDS characterized by induced levels γ-2HAX and phosphorylated ATM that correlated with AML blast percentages (Boehrer et al. Oncogene 2009). The low incidence of p53 mutations (7% in adult AML, The Cancer Genome Atlas Research Network, N Engl J Med 2013) in AML indicates downstream suppression of DNA damage control system proteins, as was proposed previously by overexpression of MDM2 and BCL-2 (Wojcik et al. Neoplasma 2005). However, the role of p53 and downstream effectors regulating the DNA damage pathway in CD34+ AML blasts is still relatively unclear.Surprisingly, using RPPA analysis, we found a common significant increase in p53 phosphorylation at serine 15 in pediatric AML as compared to CD34+ NBM (AML n=31 and NBM n=10, Mann-Whitney U, P = 0.003). Therefore, we challenged to target p53 using a shRNA approach. In the sh-p53 model, primary pediatric AML samples transduced with sh-p53 showed a significant increase in AML proliferation over time as compared to scrambled control (paired-samples t-test, P = 0.005). The overall AML cell viability was significantly improved in sh-p53 AML cultures (paired-samples t-test P = 0.001). DNA cell cycle analysis revealed that sh-p53 AML cultures contained significant increased percentages of cells in S and G2/M phases of the cell cycle as compared to their scrambled controls (paired samples t-test, P = 0.015). The CD34+ leukemic population was maintained in p53 knockdown cultures and thereby outcompeted scrambled control cultures. Knockdown of p53 in primary pediatric AML samples resulted in a higher proliferation rate, repressed differentiation and prolonged survival of AML blasts, especially in MLL-rearranged AML. Interestingly, we previously showed that MLL-rearranged AML samples express significantly higher BCL-2 peptide phosphorylation as compared to NBM (Kampen et al. Leukemia 2014). Growth factors are extrinsic cues that can regulate BCL-2 expression (Pidgeon et al. Br J Cancer 2001). Growth factors like VEGF, have been shown to be involved in leukemia pathogenesis in the bone marrow where leukemic stem cells are preserved in tightly controlled niches (Kampen et al. Cell Mol Life Sci 2013). Accordingly, we found that FGF and VEGFC stimulation of CD34+ AML blasts both significantly accelerated BCL-2 mRNA expression by 28-fold and 2-fold, indicating that indeed the niche is important for inhibiting pro-apoptotic p53-dependent signals further downstream the pathway, which protects AML CD34+ blasts (Student’s t-test, both P < 0.001), where p53 and p21 mRNA expression remained unchanged. The availability of VEGFR-2 and FGFR1 previously highlighted their therapeutic targeting potential in AML (Kampen et al. Leukemia 2006, Kentsis et al, Nature Med 2012). This study presents the effect of p53 suppression on AML blast proliferation, survival and maintenance in pediatric AML and indicates the importance of exploring extrinsic factors that contribute to DNA damage control system suppression in vivo for therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Capillary nano-immunoassay for Akt 1/2/3 and 4EBP1 phosphorylation in acute myeloid leukemia.

BackgroundOverall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse being a major cause of mortality. The PI3K-Akt-mTOR kinase pathway plays a vital role in pro-survival signals within leukemic cells and inhibition of this pathway is being investigated to improve patient outcomes. Tracking activation of multiple signaling proteins simultaneously in patient samples can be challenging especially with limiting cell numbers within rare sub-populations.MethodsThe NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have utilized this nano-immunoassay to examine activation of Akt 1/2/3 and downstream mTOR target - eukaryotic initiation factor 4E-Binding Protein 1 (4EBP1).ResultsAssays for Akt 1/2/3 and 4EBP1 were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. Target inhibition was studied using mTOR 1/2 inhibitor AZD-8055 and results were corroborated by Western blotting. The assay was able to quantify nanogram amounts of 4EBP1 and Akt 1/2/3 in AML cell lines and primary pediatric AML samples and results were quantifiable, consistent and reproducible.ConclusionOur data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this nano-immunoassay prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy.

EphB1 Downregulation In Acute Myeloid Leukemia: Suppressing p53-Dependent DNA Damage Control System

BackgroundEphrin receptors (Eph) have been implicated to contribute to cancer pathogenesis in a way by which receptors can both promote or suppress cancer progression depending on their cellular contexts. Mechanism for Eph receptor tumor suppressor functions are unknown. In a previous study, we found that pediatric acute myeloid leukemia (AML) sample lysates showed a significant decreased EphB1 peptide activity in comparison to normal bone marrow (NBM). In this study, we challenged to investigate EphB1 receptor transcriptional regulation and biological insights in AML. Materials and MethodsQuantitative RT-PCR analysis and bisulfate pyrosequencing was used to examine mRNA expression and methylation in AML cell lines and AML patient samples. AML cell proliferation (cell counts) and apoptosis (annexin V/PI) were analyzed after EfnB1 ligand stimulation. We generated constitutive EphB1 overexpression constructs in AML cell lines. Downstream in vitro effects were studied using quantitative RT-PCR, flowcytometry, phospho-proteome arrays, chromatin immunoprecipitation (ChIP), and immunoblot analysis. ResultsFirst, we examined EphB1 mRNA expression and methylation in primary pediatric AML samples, which revealed that EphB1 mRNA expression was significantly lower in unfavorable AML cytogenetic subgroups as compared to favorable AML patient samples (Fig. A, P = 0.013). The EphB1 promoter region was found increasingly methylated in approximately 20% of the pediatric AML patient samples (n=21), but not in NBM (n=5, Fig. B). In AML cell lines, we confirmed that EphB1 promoter methylation resulted in epigenetic gene silencing (P = 0.010).As a next step we challenged to study the biological relevance of EphB1 suppression. EfnB1 stimulation of EphB1high THP-1 cells inhibited the cell proliferation and induced apoptosis, while EphB1low HL60 and MOLM13 cells remained unaffected upon EfnB1 stimulation. Cell cycle analysis revealed that EfnB1 stimulation mediated phenotypic effects in EphB1high THP-1 cells could be assigned to a G2/M cell cycle arrest. The cell cycle arrest and the induction of apoptosis as a common consequence was confirmed by phospho-proteome, immunoblot, and qRT-PCR analysis. We found an induction of DNA damage control protein activation and cell cycle inhibitors Chk-2, p21, p27, p53, and CDC2tyr15, and a reduction of cell survival proteins HSP27 and BCL-2 accompanied by an increase of Bax. Next, we investigated whether EfnB1 stimulation of THP-1 could have restored the DNA damage control system by increasing DNA binding of mutant and wildtype p53. Performing ChIP assays using p53 antibodies recognizing wildtype or both mutant and wildtype p53 followed by p21 and MDM2 quantitative RT-PCR analysis revealed that EfnB1 stimulation of THP-1 cells significantly increased p53 DNA binding (Fig. C).Introducing a constitutive EphB1 overexpression into EphB1 methylated HL60 and MOLM13 cell lines resulted in an instant increase in apoptosis relatively to empty vector control cells. As expected, G2/M cell cycle inhibiting phosphorylation of CDC2tyr15 was upregulated. EfnB1 stimulation of the GFP sorted remaining viable cells showed to decrease the AML cell survival in EphB1 overexpressed AML cells as compared to empty vector controls. Additionally, we found that EphB1 demethylation by 5-Aza-2'-deoxycytidine was able to restore EphB1 membrane protein expression in EphB1 methylated AML cells and simultaneously reduced the AML cell survival. ConclusionsIn this study, we found a common downregulation of EphB1 mRNA that was significantly associated with an unfavorable prognosis in pediatric AML. The downregulation of EphB1 could be explained by EphB1 promoter hypermethylation in a subset of AML samples. Downregulation of EphB1 was found to be one of the mechanisms through which AML cells suppress p53 DNA binding that impairs the DNA damage control system to support a proliferative and anti-apoptotic AML cell survival advantage.▪ Disclosures:No relevant conflicts of interest to declare.

Abstract C200: Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia.

Abstract Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse in AML being a major cause of mortality. Risk stratification by cytogenetic and molecular aberrations has changed the prognosis for some patients, but for a majority of the patients (especially with normal cytogenetics) improvement in therapy is needed. Understanding key differences in survival pathways such as the mTOR pathway within leukemic cells could be potentially exploited to develop new drug therapy and design relapse therapy. Tracking pathway activation in patient samples is challenging in AML since samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have used this novel nano-technology based system to study the mTOR pathway in AML samples (n=8) obtained from the Children's Oncology Group (COG) Myeloid Disease Reference Laboratory. Assays for downstream mTOR target protein-eukaryotic initiation factor 4E binding protein 1 (4EBP1) were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. The assay was able to detect as little as 80 ng of protein which corresponded to 1000 cells per assay. MV4-11 cells were treated with the dual mTOR 1/2 inhibitor AZD-8055 for 24 hours and reduced phosphorylation of 4EBP1 was confirmed by Western Blotting and corroborated by the NanoPro assay. This nano-immunoassay was able to reliably quantify amounts of 4EBP1 (total and phosphorylated forms) in primary pediatric AML samples (n=8) thawed following cryopreservation. The relative luminescence units (measure of chemiluminescence and protein signal) were plotted against exposure time and the linearity of the graph provided evidence that the signal is sensitive over a range of three logs. Further, these samples were treated with AZD-8055 for 24 hours followed by analysis of changes in signaling profiles for total and phosphorylated Ser65 and Thr37/46. As expected, complete reduction of phosphorylation was noted in the treated samples (n=4). The assays obtained were quantifiable, consistent, and reproducible and enabled us to delineate activation patterns of the mTOR pathway in primary AML samples and evaluate drug efficacy for targeted agents. Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this novel nano-immunoassay based platform prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C200. Citation Format: Himalee Sabnis, Heath Bradley, Todd Cooper, Kevin Bunting. Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C200.

Anti-VEGFC Treatment Reduces the Leukemic Outgrowth of Primary CD34+ Pediatric Acute Myeloid Leukemia Cells

Abstract 1425Previously, it was demonstrated that exogenous addition of vascular endothelial growth factor C (VEGFC) increased the leukemic cell viability, reduced apoptosis via activation of Bcl-2, and decreased chemotherapy induced apoptosis via its receptor FLT-4 (Further revert to as VEGFR3) (Dias et al. Blood 2002). Furthermore, it was shown that VEGFC promotes angiogenesis by induction of COX-2 through VEGFR3 activation in THP-1 cells (Chien et al. Carcinogenesis 2005). We have previously found that endogenous VEGFC expression is associated with decreased drug responsiveness in childhood acute myeloid leukemia (AML), both in vitro as well as in vivo (de Jonge et al. Clinical Cancer Research 2008). In addition, high VEGFC mRNA expression is strongly associated with reduced complete remission and overall survival in adult as well as pediatric AML (de Jonge et al. Blood 2010). It was thought that the leukemic blast population is organized as a hierarchy, whereby leukemia initiating cells (LICs) reside at the top of this hierarchy, and it is only these cells that have the capacity to engraft in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The LIC is thought to be enriched in the CD34+ leukemic cell fraction and is shown to expand in vitro using a myeloid cytokine mix of IL-3, TPO, and G-CSF in colony forming cell (CFC) assays and long-term culture-initiating cell (LTC-IC) assays (Guan et al. Exp. Hematol. 2002, van Gosliga et al. Exp. Hematol. 2007). Moreover, LTC-IC assays performed in limiting dilution detect the in vitro outgrowth potential of stem-like cells that reside underneath the stromal cell layer. In this study, we set out to investigate the potential of anti-VEGFC treatment as an inhibitor of the outgrowth of LICs within the CD34+ fraction of primary AML samples. First, we determined the possibility of an autocrine loop for VEGFC in AML. Pediatric AML cell (n=7) derived VEGFC levels were found to be 1.4-fold increased (P =.008) compared to secreted VEGFC levels from normal bone marrow (NBM) cells (n=4). Pediatric AML blast cells showed KDR (further revert to as VEGFR2) membrane expression in 44 out of 50 patient samples (varying 8–99% of the total blast population), whereas on NBM cells VEGFR2 expression was below 5%. VEGFR3 expression was below 5% on both leukemic blasts and NBM cells. We evaluated the effect of anti-VEGFC (VGX-100, kindly provided by Vegenics, used at a concentration of 30 μg/ml) treatment on the CD34+ isolated compartment of pediatric AML bone marrow samples. Anti-VEGFC treatment reduced the outgrowth potential of AML derived CD34+ cells (n=2) with >25% in CFC assays. Interestingly, morphological analysis revealed a 3-fold enhanced formation of macrophages. LTC-IC assays demonstrated a (15% to 50%) decrease in the long-term growth of CD34+ isolated AML cells in 3 out of 4 patient samples. Morphological characterization of the suspension cells suggested a shift in development along the myelomonocytic lineage after two weeks of anti-VEGFC treatment. With FACS analysis, these cells showed a higher number of cells stained positive for CD11b, and CD14, and lower numbers where positive for CD34. Anti-VEGFC treated LTC-IC assays in limiting dilution demonstrated a (44% and 74%) reduction in the outgrowth potential of long-term cultured CD34+ isolated AML cells and blocked the erythroid colony formation in 2 out of 3 patient samples. Anti-VEGFC treatment did not have an effect on the outgrowth of CD34+ sorted NBM cells in the various assays (n=2). In conclusion, anti-VEGFC treatment of the CD34+ isolated fraction from primary pediatric AML samples showed a reduction of AML outgrowth. Differentiating cells are skewed to the myelomonocytic lineage upon anti-VEGFC treatment. We hypothesize that deprivation of VEGFC in primary CD34+ AML cell cultures results in enhanced leukemic cell death and abates an important proliferation signal for AML cells. Yet, further investigations are warranted. [Display omitted] Disclosures:Baldwin:Circadian Technologies Limited: Employment. Robert:Circadian Technologies Limited: Employment, Membership on an entity's Board of Directors or advisory committees.

Multiparameter FACS Analysis of G-CSF and IL-6 Signaling through Stat3 and Stat5 In Primary Pediatric AML Samples.

AbstractAbstract 1051Pediatric acute myeloid leukemia (AML) remains a devastating disease with a relapse rate approaching 50%. New targeted therapies, such as kinase inhibitors, are entering clinical use rapidly, yet the signaling abnormalities targeted by these drugs remain poorly understood. Pathways involving Stat3 and Stat5 are known to be aberrantly active in AML. Increased constitutive and/or ligand-activated Stat3/5 activity has been associated with chemoresistant disease, likely due to increased pro-survival gene expression. Our hypothesis is that analysis of Stat3/5 activation, and expression levels of key proteins in the pathways, will provide insight into mechanisms of aberrant signaling, and will guide the development and implementation of targeted therapies. We performed multiparameter FACS analysis of tyrosine-phosphorylated Stat3 (pStat3; n=56) and pStat5 (n=38) in pediatric AML samples from the Children's Oncology Group. We also measured expression levels of Stat3 protein, G-CSF receptor (G-CSFR), and gp130 (IL-6 receptor; n=56). Constitutive activation was common and varied over a wide range (median 36.1% pStat3+, range: 0.7 – 82%; median 33.9% pStat5+, range: 5.9 – 77%). There was non-significant correlation between the percent of pStat3+ cells and the percent of pStat5+ cells in unstimulated cells. As expected, there was a significant correlation between constitutive %pStat3+ and total Stat3 expression (by mean fluorescence intensity (MFI)), with linear correlation coefficient R=0.375 (p=0.004). Most samples were not responsive to G-CSF or IL-6. For Stat3 activation, 33/56 (59%) failed to demonstrate at least a 2-fold increase in the pStat3 MFI after 15 minute treatment with G-CSF (100 ng/ml), and 35/56 (63%) did not respond to IL-6 (50 ng/ml IL-6 + 100 ng/ml soluble IL-6 receptor (sIL6R)). A pStat3 response to these high doses only was seen in 23% of samples after G-CSF and 20% after IL-6. A pStat3 response was seen to 10 ng/ml G-CSF in 18% and to 5 ng/ml IL-6 + 10 ng/ml sIL6R in 16%. No samples responded to 1 ng/ml G-CSF and 1 sample (2%) responded to 0.5 ng/ml IL-6 + 1 ng/ml sIL6R. The pattern of pStat5 responses to G-CSF was similar: 53% did not respond to any dose, 26% responded to the highest dose, 21% responded to the intermediate dose, and none responded to the lowest dose. Only 1 sample (3%) showed an increase in pStat5 after IL-6 stimulation. As in unstimulated cells, there was no correlation between the pStat3 and pStat5 responses to G-CSF, suggesting divergence of the pathways from G-CSFR to each Stat. In contrast, the pStat3 response to G-CSF significantly correlated with the pStat3 response to IL-6 (R=0.423, p=0.001), suggesting convergence of the pathways from each receptor to Stat3. Surface G-CSFR expression positively correlated with the GCSF-induced pStat3 response (R=0.365, p=0.006). Interestingly, surface gp130 expression negatively correlated with the IL6-induced response (R=-0.352, p=0.008), meaning samples with robust IL-6 responses tended to have fewer gp130+ cells. In summary, multiparameter FACS analysis of baseline and ligand-induced Stat3 and Stat5 activity provides biologic insight into signaling aberrancies in AML, and this knowledge will promote rational design and testing of new therapies. Disclosures:No relevant conflicts of interest to declare.