Maintenance Of Acute Myeloid Leukemia Research Articles

Inhibition of the PI3K/AKT/mTOR Pathway Leads to Down-Regulation of c-Myc and Overcomes Resistance to ATRA in Acute Myeloid Leukemia

Acute Promyelocytic Leukemia (APL) accounts for 5% of all cases of acute myeloid leukemia (AML). This disease is highly curable with all-trans-retinoic acid (ATRA) based therapy. In non-APL AML, ATRA has limited activity, and little is known about mechanisms of ATRA resistance. The apparent selective efficacy of ATRA in PML/RARα-associated APL poses an important question as to whether the presence of this fusion protein renders APL uniquely susceptible. Two compelling arguments can be made to counter this view. First, experiments in vitro show that ATRA effectively differentiates HL-60 cell lines, which lack the PML/RARα fusion protein. Second, clinical studies with ATRA in previously untreated older AML patients (excluding APL) have reported clinical activity. These observations confirm the therapeutic potential of ATRA beyond APL. In this context, our group has previously identified the lysine demethylase LSD-1, as a therapeutic target to re-sensitize leukemic blasts to ATRA. A clinical investigation of ATRA combined with LSD-1 inhibition is currently underway (NCT02273102). It is likely that other defects leading to ATRA resistance will be similarly amenable to pharmacologic manipulation. Defects in the proto-oncogene c-Myc have been widely implicated in the initiation and maintenance of AML. Over-expression of c-Myc in leukemic blasts enhances clonogenic survival and blocks ATRA induced differentiation. We hypothesized that down-regulation of c-Myc might increase the anti-leukemic effects of ATRA in AML. To date, c-Myc has been an evasive target for direct pharmacologic inhibition however, inhibitors of the PI3K/AKT/mTOR pathway have been shown to indirectly lower levels of c-Myc in leukemic blasts.In the current study, we show that the pro-differentiation effects of ATRA are markedly potentiated when combined with agents that target PI3K/AKT/mTOR signalling. In AML cell lines and primary patient samples, we observed additive pro-differentiation effects when ATRA was combined with inhibitors of PI3K (ZSTK474) and mTOR complex proteins (Torin-1, WYE-125132). However, when combined with the bromodomain inhibitor NVP-BEZ235, a dual inhibitor of PI3K and mTOR, we observed synergistic induction of CD11b by FACS analysis. Combination studies revealed loss of cell viability, cell cycle arrest in G1 phase, and impaired clonogenic survival, which was more prominent for ATRA combination treatments than with any agent used alone (Figure 1). To assess the role of c-Myc in mediating these effects, we measured c-Myc protein levels and PI3K/AKt/mTOR pathway markers at different time-points following treatment with ATRA alone and in combination with the inhibitors described above (Figure 2). Our findings suggest that ATRA alone quickly down-regulates c-Myc (within 6 hours) through transcriptional repression. Disruption of the PI3K/AKT/mTOR pathway further down-regulates c-Myc (within 3 hours) through destabilization and enhanced degradation. ATRA combined with NVP-BEZ235 produced maximal c-Myc suppression, and led to more cell kill than any other combination tested. Detailed analysis of changes in the transcriptome in MV-411 cells following treatment with ATRA and NVP-BEZ235 revealed that both agents act jointly on the regulation of the same biological pathways and processes, but regulate different sets of genes within these pathways. Updated mechanism based studies will be presented.In conclusion, suppression of c-Myc levels through disruption of PI3K/AKT/mTOR signalling augments the anti-leukemic effects of ATRA. These data support the clinical investigation of ATRA combined with rapalogs or bromodomain inhibitors. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Contribution of Bone Marrow Stromal Cells to Leukemia Evolution: Differential Support of Leukemia Cell Growth at Diagnosis and Remission

Introduction: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of the hematopoietic progenitor cells characterized by excessive proliferation of early progenitor cells in the bone marrow (BM) and is hence associated with a variable prognosis. BM niches are specialized types of microenvironment that actively support the growth, maturation and maintenance of hematopoietic stem and progenitor cells. They include a complex network of extracellular matrix proteins, soluble growth factors, and cytokines. Little is known about the leukemic microenvironment or its clinical relevance to AML initiation and propagation. The aim of the study was to define the role of stromal cells in support of leukemic cell growth in active disease as compared to the remission (Rm) state and normal stoma. Thus, we have investigated the expression of various cytokines secreted by stromal cells and involved in hematopoietic progenitor maintenance and proliferation at disease diagnosis (Dx) and Rm. Additionally, we have characterized the growth pattern of leukemic cells on various stroma cells from different sources and disease stages.Methods: Leukemic and stromal cells from BM of 4 patients with AML were collected at Dx. Stromal cells were also collected in Rm. Leukemic cells were enriched for CD34+ using magnetic beads. Patients' own stroma (POS) obtained both at Dx and Rm and the HS-5 cell line (control) were seeded on gelatin-treated plates and were grown with MesenCult medium for 42 days. Morphologic and growth rate changes were monitored every week using inverted microscopy X20. Upon adherence of 40-50% of the stromal cells, CD34+ enriched leukemic cells derived at Dx were seeded on top of POS from both Dx and Rm and were grown together for additional 2 weeks. Co-cultures were harvested and RNA was extracted followed by RT-PCR to amplify the following hematopoietic regulating genes: hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and CXCL12. The results were expressed as arbitrary units normalized to GAPDH gene expression.Results: Expression of CXCL12 and HGF was elevated in a co-culture of leukemic cells and POS obtained at Dx compared to POS from Rm in all 4 patients. Additionally, the co-culture of leukemic cells and POS from Rm showed a higher expression of CXCL12 and HGF as compared to POS alone derived in Rm (Fig. 1). Mean values of cytokine expression were 37.33, 26.44 and 38.62 at Dx and 29.83, 25.55 and 24.55 in Rm for HGF, FGF and CXCL12, respectively. Additionally, a preferential growth advantage of colonies derived from single leukemic cells was observed when these cells were seeded on POS from Dx and relapse (Rx) compared to Rm (Fig. 2A). Moreover, colonies grew mainly in a co-culture with POS while no growth was noted on healthy stromal cells or stroma derived from a patient with myeloma (Fig. 2A) or another AML patient (Fig. 2B).Conclusions: Co-culture of AML cells and POS derived at Dx show a higher expression of CXCL12 and HGF compared to POS obtained in Rm, suggesting favorable microenvironment conditions supporting AML initiation and maintenance. A synergistic effect on expression of CXCL12 and HGF was observed in a co-culture of POS with leukemic cells as compared to POS or cell-line alone. Leukemic cells show preferential growth on POS and no growth on various malignant and normal stromal cells. Leukemic cell growth was diminished in Rm compared to Dx and Rx. Studying a larger number of patients as well as investigating the specific interaction of AML cells with stromal cells derived from various disease stages is needed to confirm the above results. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NSD3-Short Is an Adaptor Protein that Couples BRD4 to the CHD8 Chromatin Remodeler

The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in acute myeloid leukemia (AML). Here, we demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3that lacks the methyltransferase domain. We show that NSD3-short is an adaptor protein that sustains leukemia by linking BRD4 to the CHD8 chromatin remodeler, by using a PWWP chromatin reader module, and by employing an acidic transactivation domain. Genetic targeting of NSD3 or CHD8 mimics the phenotypic and transcriptional effects ofBRD4 inhibition. Furthermore, BRD4, NSD3, and CHD8 colocalize across the AML genome, and each is released from super-enhancer regions upon chemical inhibition of BET bromodomains. These findings suggest that BET inhibitors exert therapeutic effects in leukemia by evicting BRD4-NSD3-CHD8 complexes from chromatin to suppress transcription.

An international phase 3 randomized, placebo-controlled study of CC-486 (oral azacitidine) maintenance therapy in patients with acute myeloid leukemia (AML) in complete remission (CR): The Quazar AML maintenance trial.

TPS7097 Background: Few inroads have been made in the past 30 years to improve overall survival (OS) in older patients (pts) with AML. While induction chemotherapy (IC) can induce CR in 40-50% of o...

Epigenetic regulators as promising therapeutic targets in acute myeloid leukemia.

Acute myeloid leukemia (AML), the most prevalent acute leukemia in adults, is an aggressive hematological malignancy arising in hematopoietic stem and progenitor cells. With the exception of a few specific AML subtypes, the mainstays of treatment have not significantly changed over the last 20 years, and are still based on standard cytotoxic chemotherapy. As a result, clinical outcome remains poor for the majority of patients, with overall long-term survival in the region of 20-30%. Recent successes in characterizing the genetic landscape of AML have highlighted that, despite its heterogeneity, many cases of AML carry recurrent mutations in genes encoding epigenetic regulators. Transcriptional dysregulation and altered epigenetic function have therefore emerged as exciting areas in AML research and it is becoming increasingly clear that epigenetic dysfunction is central to leukemogenesis in AML. This has subsequently paved the way for the development of epigenetically targeted therapies. In this review, we will discuss the most recent advances in our understanding of the role of epigenetic dysregulation in AML pathobiology. We will particularly focus on those altered epigenetic programs that have been shown to be central to the development and maintenance of AML in preclinical models. We will discuss the recent development of therapeutics specifically targeting these key epigenetic programs in AML, describe their mechanism of action and present their current clinical development. Finally, we will discuss the opportunities presented by epigenetically targeted therapy in AML and will highlight future challenges ahead for the AML community, to ensure that these novel therapeutics are optimally translated into clinical practice and result in clinical improvement for AML patients.

The epigenetic regulators CBP and p300 facilitate leukemogenesis and represent therapeutic targets in acute myeloid leukemia.

Growing evidence links abnormal epigenetic control to the development of hematological malignancies. Accordingly, inhibition of epigenetic regulators is emerging as a promising therapeutic strategy. The acetylation status of lysine residues in histone tails is one of a number of epigenetic post-translational modifications that alter DNA-templated processes, such as transcription, to facilitate malignant transformation. Although histone deacetylases are already being clinically targeted, the role of histone lysine acetyltransferases (KAT) in malignancy is less well characterized. We chose to study this question in the context of acute myeloid leukemia (AML), where, using in vitro and in vivo genetic ablation and knockdown experiments in murine models, we demonstrate a role for the epigenetic regulators CBP and p300 in the induction and maintenance of AML. Furthermore, using selective small molecule inhibitors of their lysine acetyltransferase activity, we validate CBP/p300 as therapeutic targets in vitro across a wide range of human AML subtypes. We proceed to show that growth retardation occurs through the induction of transcriptional changes that induce apoptosis and cell-cycle arrest in leukemia cells and finally demonstrate the efficacy of the KAT inhibitors in decreasing clonogenic growth of primary AML patient samples. Taken together, these data suggest that CBP/p300 are promising therapeutic targets across multiple subtypes in AML.

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.

Syk and Btk Transduce Survival and Proliferation-Inducing Signals By Activating Distinct Signaling Pathways and Transcriptional Programs in AML Cells

Acute myeloid leukemia (AML) is a genetically heterogeneous disease where multiple driver mutations coincide in branching hematopoietic subclones leading to malignant transformation. One important class of mutations alters the function of signaling intermediates such as FLT3, thereby helping AML cells to overcome the physiological communication with their microenvironment. To investigate, if also non-mutated signaling molecules might be potential drug targets in AML, we performed an unbiased proteomic kinase activity profiling. Our (phospho)proteomic and functional analyses revealed spleen tyrosine kinase (SYK) and Bruton’s tyrosine kinase (BTK) as novel non-mutated drug targets in AML. By immunohistochemical analyses we identified expression of both kinases in more than 70% of analyzed AML cases. Oncogenic SYK signaling in AML cells turned out to be critically regulated by beta-integrins upon intercellular contact formation between AML cells and bone marrow stroma cells. BTK, however, turned out to act downstream of FLT3-ITD to activate MYC-dependent oncogenic transcriptional programs. FLT3-ITD expression induced activation of BTK in cell culture models and patient-derived AML cultures and sensitized them towards BTK inhibitors. This newly identified FLT3-ITD/BTK signaling axis can be therapeutically exploited by combining FLT3 and BTK inhibitors resulting in additive apoptosis-inducing effects. As in some cases also FLT3-ITD-negative AML cells showed response to BTK inhibition, we characterized the BTK interactome and its downstream signaling networks by quantitative proteomic techniques and identified the apoptosis sensor Toll-like receptor 9 (TLR9) as an upstream activator of BTK in FLT-ITD-negative cells. In the absence of FLT3-ITD, TLR9 activates BTK to induce cell survival through NFkB as revealed by transcriptome sequencing and cell biological studies. While TLR9 stimulation enhanced AML cell proliferation, knock-down of TLR9 induced a partial cell cycle arrest and apoptosis. Thus, we here describe a novel microenvironment-derived signaling axis that may represent an important mechanism of AML maintenance in various important clinical settings including efficient FLT3-ITD inhibition and cytotoxic therapy. Taken together, these data highlight the importance of interactions between AML cells and their microenvironment and unravel cooperative dependencies between mutant oncoproteins and wild-type kinases in AML pathogenesis. They also provide a rationale for the clinical evaluation of SYK- and BTK inhibitors in AML. DisclosuresBeck:Chronic Biomedical: Employment. Schuetz:Chronix Biomedical: Employment.

GNA13, a Novel Component of the GPR84/Beta-Catenin Signaling Axis in AML Stem Cells, Regulates the Maintenance of MLLAF9 Leukemogenesis

Beta-catenin is required for the establishment of leukemic stem cells (LSC) in mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML). Targeted inhibition of beta-catenin signaling has been hampered by the lack of pathway components amenable to pharmacological inhibition. Our recent study has identified a new beta-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. Since no GPR84-specific anti-cancer compound has been developed to date, we aim to further delineate the GPR84/beta-catenin axis in order to inhibit other components of this signaling pathway pharmacologically.Preliminary data from our lab has implicated GNA13 as a potential downstream target gene of GPR84. In this study we sought to further examine the involvement of GNA13 in the GPR84/beta-catenin signaling axis. Functional studies showed that shRNA-mediated ablation of GNA13 significantly decreased the capacity of MLLAF9-LSC to form colonies in vitro (P=0.0184). We further demonstrated that GNA13 inhibition in LSC induced a significant G1 cell cycle arrest in vitro (P=0.0192). Subsequent in vivo studies showed that GNA13 downregulation in MLLAF9-LSC significantly delayed the onset of AML (P=0.0016) and significantly decreased LSC proliferation (P=0.0066). Hence GNA13 inhibition mimics the previously described GPR84-deficient phenotype in MLLAF9-LSC (Blood 2013;122:3781). In contrast, the GPR84-deficient phenotype could be rescued in MLLAF9-LSC by GNA13 overexpression in vitro (P=0.0130) and in vivo (P=0.0012). Further analysis showed that there was a significant increase in the mitotic index of GPR84-deficient LSC transduced with GNA13 cDNA in vitro, evidenced by an increase in G2/M-phase accumulation (P=0.0030) and p-histone H3 (P=0.0040). In addition, our data demonstrated a significant increase in BrdU incorporation of GPR84-deficient LSC overexpressing GNA13 in vivo (P<0.0001).In conclusion, our studies have identified GNA13, a novel component of the GPR84/beta-catenin axis. Since this pathway is pivotal for MLLAF9 AML maintenance by sustaining LSC self-renewal, targeting the GPR84/GNA13/beta-catenin signaling axis may provide a novel and promising strategy for improving the therapy and outcome of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Dose Dependent Role of Gfi1 in Human MDS and AML and Its Suitability As a Novel Target

Acute myeloid Leukemia (AML) characteristically involves a block in hematopoietic stem cell (HSC) differentiation to mature cells. This results in blast accumulation in the bone marrow (BM). Myelodysplastic syndrome (MDS) is also characterized by disturbed differentiation, but with less severe blockage of lineages. MDS often progresses to AML. Differentiation of HSC to myeloid cells is regulated by transcription factors like growth factor independence 1 (GFI1) on chromosome 1. Understanding is needed to develop AML/MDS treatments that target such factor.We investigated the role of different Gfi1 levels on the initiation, progression and prognosis of AML and whether Gfi1 could serve as a new therapeutic target of AML therapy. First, analysis of published expression array data showed that Gfi1 is expressed at a lower level in blast cells compared to control cells. In addition low Gfi1 expression level in blast cells of patients was associated with an inferior prognosis (9 months median event free survival) compared to patients with higher Gfi1 expression (32 months). With regard to MDS patients, deletion or lower levels of Gfi1 were associated with higher level of blasts.We found that in low Gfi1 expressing blasts Gfi1 target genes are up-regulated, confirming a role of Gfi1 in leukemia development. To verify that low levels or deletion of one Gfi1 allele causes AML we used mice expressing Gfi1 at the following levels: Gfi1 deficient mice (Gfi1 KO) with no expression; mice expressing Gfi1 only at 20% of WT Gfi1 expression level (Gfi1 KD); mice heterozygous for Gfi1 (Gfi1 het) with ~50% of WT expression, and WT mice. These mice were crossed with Nup98HoxD13 mice that recapitulate MDS in humans. Nup98HoxD13 tg mice develop pancytopenia and some mice develop AML or ALL. We observed that Gfi1 KD (n=13) accelerated AML development compared to Gfi1 WT mice (n=21). Complete loss of Gfi1 (n=14) inhibited AML development (p=0.03). Furthermore, deletion of one Gfi1 allele (Gfi1 het) mice (n= 26) shortened leukemia free survival (p=0.024) compared to WT mice with a higher AML-incidence of 50% whereas WT (n=46) mice have only 31%. We then confirmed our findings in an independent AML-mouse model: we crossed WT (n=31) and Gfi1-KD (n=24) mice with MLL-AF9 transgenic mice and found marginally (p=0.06) decreased leukemia free survival in Gfi1-KD mice.It has been shown that retroviral expression of onco-fusion proteins such as MLL-AF9 or AML1-Eto9a can cause AML in mice. To confirm our findings, we transplanted Gfi1 KD and WT MLL-AF9 transduced Lin-BM cells into lethally irradiated recipient mice. Mice transplanted with MLL-AF9-Gfi1 KD cells (n=7) developed AML faster than mice transplanted with MLL-AF9-WT cells (n=12; p< 0.0001). In contrast to Gfi1 KD, Gfi1 KO prevented leukemia initiation in a retroviral AML mouse model.If Gfi1 KD accelerates the emergence of AML, Gfi1 overexpression should hamper the development of AML. We thus retrovirally co-transduced, haematopoietic progenitor cells from WT mice with retroviruses overexpressing onco-fusion proteins and either Gfi1 or an empty vector. We sed the cells in liquid culture and methylcellulose. Overexpression of Gfi1 was associated with 4-fold fewer colonies and total cells relative to cells transduced with empty vector (p=0.01).In summary, either complete loss of Gfi1 or overexpression could be use as an approach to treat leukemia. To examine the role of different Gfi1 levels in AML maintenance in-vivo, we transplanted leukemic Gfi1 conditional (Gfi1 expression can be conditionally deleted) and Gfi1 up-regulation conditional (Gfi1-overexpression can be induced) cells into irradiated mice. Induced up-regulation of Gfi1 led to terminal differentiation of blast cells whereas induced loss of Gfi1 led to complete absence of blasts due to increased apoptosis.Analyzing different leukemic samples expressing Gfi1 at different level using RNA-Seq, we observed that, at low levels, Gfi1 inhibits apoptosis by inhibiting p53, at medium levels it also represses oncogenes, and at high levels it induces differentiation. Thus Gfi1 plays a dose-dependent role in AML and either very high expression or ablation of Gfi1 could be a useful novel therapeutic objective. We are currently investigating these effects on human cells. DisclosuresNo relevant conflicts of interest to declare.

Pathogenic microRNA's in myeloid malignancies.

Recent studies have significantly improved our understanding of the role microRNAs (miRNAs) play in regulating normal hematopoiesis. miRNAs are critical for maintaining hematopoietic stem cell function and the development of mature progeny. Thus, perhaps it is not surprising that miRNAs serve as oncogenes and tumor suppressors in hematologic malignancies arising from hematopoietic stem and progenitor cells, such as the myeloid disorders. A number of studies have extensively documented the widespread dysregulation of miRNA expression in human acute myeloid leukemia (AML), inspiring numerous explorations of the functional role of miRNAs in myeloid leukemogenesis. While these investigations have confirmed that a large number of miRNAs exhibit altered expression in AML, only a small fraction has been confirmed as functional mediators of AML development or maintenance. Herein, we summarize the miRNAs for which strong experimental evidence supports their functional roles in AML pathogenesis. We also discuss the implications of these studies on the development of miRNA-directed therapies in AML.

Targeting BAG-1: A novel strategy to increase drug efficacy in acute myeloid leukemia

Overexpression of antiapoptotic proteins occurs frequently in cancer, resulting in defective apoptosis that may contribute to a poor chemosensitivity of tumor cells. B-cell lymphoma (BCL) 2-associated AthanoGene-1 (BAG-1) is a prosurvival chaperone recently found involved in the maintenance of acute myeloid leukemia (AML) cells survival in vitro. Here we reported BAG-1 upregulation in 87 of 99 analyzed AML patients with respect to healthy control samples applying reverse phase protein assay. Silencing of BAG-1 expression confirmed a decreased BCL-2 protein level but, in addition, provoked the increased transcription of GADD34 stress sensor. Furthermore, a dephosphorylation of eIF2α, as well as alteration of expression of IRE-1 and CHOP proteins, were documented, suggesting that a disruption of the endoplasmic reticulum stress/unfolded protein response was provoked by downregulation of BAG-1. A similar phenomenon was triggered after addition of Thioflavin S, which was shown to block BAG-1/BCL-2 interaction and to increase cell death, enforcing a prosurvival role of the BAG-1 protein in AML. Interestingly, synergic cytotoxic effects of doxorubicin, VP16 drugs, and ABT-737 compound were observed when Thioflavin S was coupled with these drugs. Taken together, our results gave further proof that upregulation of BAG-1 plays a critical role in AML and that BAG-1 targeting might be considered for a combined therapeutic strategy with conventional chemotherapy drugs in the treatment of AML patients.

TGIF1 is a negative regulator of MLL-rearranged acute myeloid leukemia.

Members of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing transcription factors are important downstream effectors of oncogenic fusion proteins involving the mixed lineage leukemia (MLL) gene. A well-characterized member of this protein family is MEIS1, which orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general importance of this family in malignant hematopoiesis, we therefore tested the potential function of TGIF1 in the maintenance of MLL-rearranged AML. Gene expression analysis of MLL-rearranged patient blasts demonstrated reduced TGIF1 levels, and, in accordance, we find that forced expression of TGIF1 in MLL-AF9-transformed cells promoted differentiation and cell cycle exit in vitro, and delayed leukemic onset in vivo. Mechanistically, we show that TGIF1 interferes with a MEIS1-dependent transcriptional program by associating with MEIS1-bound regions in a competitive manner and that the MEIS1:TGIF1 ratio influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML.

Epigenetic modifications of splicing factor genes in myelodysplastic syndromes and acute myeloid leukemia.

Somatic mutations in splicing factor genes have frequently been reported in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Although aberrant epigenetic changes are frequently implicated in blood cancers, their direct role in suppressing one or both alleles of critical splicing factors has not been previously examined. Here, we examined promoter DNA hypermethylation of nine splicing factors, SF3B1, SRSF2, U2AF1, ZRSR2, SF3A1, HNRNPR, MATR3, ZFR, and YBX3 in 10 leukemic cell lines and 94 MDS or AML patient samples from the Australasian Leukemia and Lymphoma Group Tissue Bank. The only evidence of epigenetic effects was hypermethylation of the YBX3 promoter in U937 cells in conjunction with an enrichment of histone marks associated with gene silencing. In silico analysis of DNA methylation data for 173 AML samples generated by the Cancer Genome Atlas Research Network revealed promoter hypermethylation of the gene encoding Y box binding protein 3, YBX3, in 11/173 (6.4%) AML cases, which was significantly associated with reduced mRNA expression (P < 0.0001). Hypermethylation of the ZRSR2 promoter was also detected in 7/173 (4%) cases but was not associated with decreased mRNA expression (P = 0.1204). Hypermethylation was absent at the promoter of seven other splicing factor genes in all cell lines and patient samples examined. We conclude that DNA hypermethylation does not frequently silence splicing factors in MDS and AML. However, in the case of YBX3, promoter hypermethylation-induced downregulation may contribute to the pathogenesis or maintenance of AML.

Regulation of Trib2 by an E2F1-C/EBPα feedback loop in AML cell proliferation

AbstractThe loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein α (C/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop for E2F1, C/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C/EBPα-p42, and in normal granulocyte/macrophage progenitor cells, we detect C/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle or Trib2 knockdown resulted in a block in AML cell proliferation. Our work proposes a novel paradigm whereby E2F1 plays a key role in the regulation of Trib2 expression important for AML cell proliferation control. Importantly, we identify the contribution of dysregulated C/EBPα and E2F1 to elevated Trib2 expression and leukemic cell survival, which likely contributes to the initiation and maintenance of AML and may have significant implications for normal and malignant hematopoiesis.

ZFX Controls Propagation and Prevents Differentiation of Acute T-Lymphoblastic and Myeloid Leukemia

Tumor-propagating cells in acute leukemia maintain a stem/progenitor-like immature phenotype and proliferative capacity. Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) originate from different lineages through distinct oncogenic events such as MLL fusions and Notch signaling, respectively. We found that Zfx, a transcription factor that controls hematopoietic stem cell self-renewal, controls the initiation and maintenance of AML caused by MLL-AF9 fusion and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx prevents differentiation and activates gene sets characteristic of immature cells of the respective lineages. In addition, endogenous Zfx contributes to gene induction and transformation by Myc overexpression in myeloid progenitors. Key Zfx target genes include the mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescues the propagation of Zfx-deficient AML. These results show that distinct leukemia types maintain their undifferentiated phenotype and self-renewal by exploiting a common stem-cell-related genetic regulator.

The Epigenetic Regulators CBP and p300 Facilitate Leukemogenesis and Represent Therapeutic Targets In Acute Myeloid Leukemia (AML)

Although molecularly and clinically heterogeneous, AML is characterised by aberrant transcription and abnormal epigenetic regulation. The transcriptional co-activators CBP and p300 have well characterised roles in hematopoiesis and hematopoietic stem cell (HSC) function. These are mutated in AML and also bind a number of AML-associated oncogenes. However, despite this, their roles in the induction and maintenance of AML are poorly understood. To address this question, we have combined genetic and pharmacological inhibition of CBP and p300 in AML and normal hematopoiesis.Using a murine model where Cbp was conditionally deleted from murine hematopoietic stem and progenitor cells (HSPC), either prior to, or following expression of a number of AML-associated oncogenes, we assessed the role of Cbp in the induction and maintenance of AML. We demonstrated that although not an absolute requirement, Cbp confers a selective advantage for robust immortalisation in vitro, and that Cbp is also an important requirement for the generation and maintenance of AML in vivo. Furthermore, redundancy between Cbp and p300 in myeloid transformation was demonstrated, as p300 knockdown further decreased proliferation in Cbp-/- AML cells.We next validated CBP/p300 as potential therapeutic targets, using a pharmacological strategy. Using a selective small molecule inhibitor (C646) of the lysine acteyltranferase (KAT) activity of CBP/p300, we demonstrated a significant decrease in growth and clonogenic potential across multiple AML subtypes in vitro. This was mediated through induction of apoptosis and cell cycle arrest. Importantly, no alteration in the growth of normal murine and human hematopoietic progenitors was detected at similar doses. We further demonstrated that inhibition of CBP/p300 KAT activity in human leukemia cells alters a transcriptional programme associated with genomic integrity, linking transcriptional changes to the cellular phenotype. Finally, we demonstrated the efficacy of the HAT inhibitors to decrease clonogenic growth across a panel of primary AML patient samples, representing multiple genetic subtypes. Taken together, these data suggest that CBP/p300 are involved in leukemogenesis across multiple subtypes in AML and that targeting these proteins may be possible with an acceptable side-effect profile. Disclosures:No relevant conflicts of interest to declare.

Parallel Transcriptional Analysis of Multiple Stem and Progenitor Populations Identifies Novel Commonly Dysregulated and Functionally Relevant Targets in AML

AbstractAbstract 1875Recent experimental evidence suggests that acute myeloid leukemia (AML) originates from hematopoietic stem and progenitor cells (HSPC) following the acquisition of multiple genetic or epigenetic changes that initially give rise to pre-leukemic HSPC (pre-LSC) and then to fully transformed leukemia stem cells (LSC). Relapse continues to be the major cause of death in most subtypes of AML, suggesting that current therapies are largely ineffective in eliminating LSC and pre-LSC. Cellular heterogeneity and the recent observation that LSC are contained within different phenotypic cellular compartments are challenges for the identification of pathways contributing to the initiation and maintenance of AML. To address these challenges we employed a novel strategy of parallel transcriptional analysis of multiple phenotypic HSPC populations from individuals with AML with normal karyotype (N=5), -7/7q- (N=6) and complex karyotype (N=5), including long-term HSC, short-term HSC, and granulocyte-monocyte progenitors (GMP), and comparison to corresponding cell populations from age-matched healthy controls (HC) (N=6). Specifically, we sorted Lin−/CD34+/CD38−/CD90+ (LT-HSC), Lin−/CD34+/CD38−/CD90− (ST-HSC), and Lin−/CD34+/CD38+/CD123+/CD45RA+ (GMP), and hybridized RNA to Affymetrix GeneST 1.0 expression arrays. Differential gene expression was determined within each compartment by direct comparison of AML LT-HSC vs. HC LT-HSC, AML ST-HSC vs. HC ST-HSC, and AML GMP vs. HC GMP. Subsequent intersection of all differentially expressed genes revealed that only a relatively small number of 6 to 11 genes were consistently dysregulated in all examined leukemic stem and progenitor cell compartments.Interleukin 1 receptor accessory protein (IL1RAP) was one of the most significantly upregulated genes in LT-HSC, ST-HSC, and GMP in all examined subtypes of AML. IL1RAP is a transmembrane protein required for signaling through several receptors of the IL1 family, including IL-1R1 and ST2. We detected significant overexpression of IL1RAP protein on HSC and progenitor cells of AML patients. Interestingly, CD34+/Lin+ precursor cells showed only a marginal increase of IL1RAP at the protein level in AML, underscoring the importance of purifying HSPC with stringent lineage depletion. We performed fluorescence in situ hybridization in sorted IL1RAP+ and IL1RAP− cells from -7 AML. We observed that the -7 clone was restricted to IL1RAP+ cells, while IL1RAP- cells did not display monosomy 7, demonstrating that IL1RAP overexpression is a distinguishing feature of the cells of the -7 clone.Patients with normal karyotype AML showed a wider range of IL1RAP expression levels; some were as high as in -7 AML and others were as low as in HC. We asked whether IL1RAP expression levels were associated with known clinical or molecular parameters. We analyzed two published datasets of patients with normal karyotype AML (Metzeler, Blood. 2008;112:4193–4201; Tomasson, Blood. 2008;111:4797–4808). Patients with high IL1RAP levels showed inferior overall survival than patients with lower IL1RAP (p=2.2×10−7; median survival: 7.82 mo. for IL1RAP high, 20 mo. for IL1RAP low). Multivariate analysis using a Cox regression model showed that high IL1RAP status was an independent prognostic factor (p=0.002), and even stronger than FLT3 mutation status (p=0.006). In addition, we analyzed data from 183 patients with MDS and found IL1RAP expression to be specifically elevated in cases with RAEB-2, suggesting a role of IL1RAP in MDS and in the progression to AML.Downregulation of IL1RAP protein expression in 4 AML cell lines (THP1, OCI-AML3, HL60, HEL) led to a significant 45–98% decrease in clonogenic growth and increased apoptosis in vitro. To assess the effects of IL1RAP downregulation in vivo, we performed xenotransplants into immunodeficient NOD/SCID/IL2Rg-null mice. THP-1 AML cells showed a 92% reduced proliferation and infiltration of hematopoietic organs upon IL1RAP knockdown in comparison to a non-silencing control in vivo. Genetic studies to assess the role of IL1RAP in the initiation and maintenance of AML in an IL1RAP−/− mouse model are currently ongoing.In summary, our study provides a map of consistently dysregulated transcripts across multiple fractionated stem and progenitor cell types from patients with AML, and identifies IL1RAP as a putative new therapeutic and prognostic target in stem cells in AML and MDS. Disclosures:No relevant conflicts of interest to declare.

FLT3-ITD+ AML Stem/Progenitor Cells Demonstrate Increased SIRT1 Expression and Enhanced Sensitivity to SIRT1 Inhibition

Abstract 1368In acute myeloid leukemia (AML) small populations of leukemia stem and progenitor cells are resistant to available therapies and represent an important barrier to response and cure. There is pressing need to develop new treatment approaches to target AML stem/progenitor cells. The NAD-dependent deacetylase SIRT1 plays an important role in protection of stem cells from stress. We have recently shown that SIRT1 inhibition enhances elimination of CML LSC via acetylation and activation of p53 (Cancer Cell 2012, 21:266). Here we evaluated the role of SIRT1 in growth and maintenance of AML stem/progenitor cells. We observed higher levels of SIRT1 expression in AML compared to normal CD34+CD38− and CD34+CD38+ cell, with highest expression levels in samples from patients with poor-risk cytogenetic abnormalities. We tested the effects of the SIRT1 inhibitor Tenovin-6 (TV-6) on CD34+ cells from high-risk AML patients (n=12) and healthy controls (n=4). AML CD34+ cells demonstrated varying degrees of sensitivity to TV-6, but TV-6 treatment did not significantly reduce viability of AML CD34+ cells taken as a group, compared to normal cells (p=0.8). However, we observed significantly reduced viability of a subset of samples bearing the FLT3-ITD mutation following TV-6 treatment (p=0.01). We therefore tested the effects of TV-6 on a larger set of poor risk AML samples with the FLT3-ITD mutation (n=11), or wild-type FLT3 (FLT3-WT, n=11). The FLT3-ITD mutation was associated with significantly enhanced sensitivity of AML CD34+ cells to TV-6 induced apoptosis (LD50 for FLT3-ITD=1.9μM; FLT3-WT=3.9μM, p=0.009), and inhibition of cell growth measured using the CellTitre Glo luminescent cell viability assay (FLT3-ITD IC50=0.73μM; versus FLT3-WT IC50=1.4μM, p=0.01). Therefore the presence of the FLT3-ITD mutation identifies a subset of AML samples that are very sensitive to SIRT1 inhibition. Tenovin-6 treatment increased acetylated as well as total p53 levels in FLT3-ITD+ AML CD34+ cells. Tenovin-6 also induced apoptosis and inhibited growth of the FLT3-ITD+ and p53 wild type cell line MV4-11. SIRT1 knock-down reduced TV-6-induced apoptosis and growth inhibition in MV4-11 cells confirming that TV-6 effects are related to SIRT1 inhibition. The growth inhibitory and pro-apoptotic effects of TV-6 in MV4-11 cells were also significantly reduced by p53 knock-down (TV-6 1μM, p53 shRNA 39±8% inhibition, control ShRNA 66±2% inhibition, p=0.04, n=3). Taken together these results support a potential role for p53 activation in mediating the effects of TV-6 treatment in FLT3-ITD+ AML cells. Sequencing studies indicated that only one sample in the FLT3-WT group harbored a p53 mutation, suggesting that their resistance to SIRT1 inhibitors is related to mechanisms other than p53 mutation. Flow cytometry analysis demonstrated increased SIRT1 levels in FLT3-ITD (n=7) compared to FLT3-WT (n=9) AML CD34+ cells (p=0.01). We used lentivirus vectors to ectopically express a FLT3-ITD construct in human cord blood CD34+ cells. Expression of FLT3-ITD resulted in significantly increased SIRT1 expression levels compared to FLT3-WT or vector controls. Consistent with results obtained with samples from AML patients, FLT3-ITD transformed CB CD34+ cells demonstrated enhanced sensitivity to TV-6 mediated apoptosis (TV6 1μM, FLT3-ITD 44±3% survival, n=5; FLT3-WT 59±2% survival, n=5, p=0.04; empty vector 64±4% survival, p=0.02, n=5) and cell growth inhibition. These results indicate that FLT3-ITD-mediated SIRT1 overexpression may contribute to the increased sensitivity of FLT3-ITD AML CD34+ cells to SIRT1 inhibition. In conclusion, our studies indicate that AML stem/progenitor cells demonstrate varying degrees of sensitivity to SIRT1 inhibition reflecting underlying genetic lesions. Importantly the poor prognosis FLT3-ITD mutation is associated with increased SIRT1 expression and enhanced sensitivity to SIRT1 inhibition. In view of the limited success of small molecule FLT3 inhibitors so far, the use of SIRT1 inhibitors may offer an attractive approach to target FLT3-ITD+ AML stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Overexpression of IL-1 receptor accessory protein in stem and progenitor cells and outcome correlation in AML and MDS

AbstractCellular and interpatient heterogeneity and the involvement of different stem and progenitor compartments in leukemogenesis are challenges for the identification of common pathways contributing to the initiation and maintenance of acute myeloid leukemia (AML). Here we used a strategy of parallel transcriptional analysis of phenotypic long-term hematopoietic stem cells (HSCs), short-term HSCs, and granulocyte-monocyte progenitors from individuals with high-risk (−7/7q−) AML and compared them with the corresponding cell populations from healthy controls. This analysis revealed dysregulated expression of 11 genes, including IL-1 receptor accessory protein (IL1RAP), in all leukemic stem and progenitor cell compartments. IL1RAP protein was found to be overexpressed on the surface of HSCs of AML patients, and marked cells with the −7/7q− anomaly. IL1RAP was also overexpressed on HSCs of patients with normal karyotype AML and high-risk myelodysplastic syndrome, suggesting a pervasive role in different disease subtypes. High IL1RAP expression was independently associated with poor overall survival in 3 independent cohorts of AML patients (P = 2.2 × 10−7). Knockdown of IL1RAP decreased clonogenicity and increased cell death of AML cells. Our study identified genes dysregulated in stem and progenitor cells in −7/7q− AML, and suggests that IL1RAP may be a promising therapeutic and prognostic target in AML and high-risk myelodysplastic syndrome.