Primary Acute Myelogenous Leukemia Research Articles

WTAP is a novel oncogenic protein in acute myeloid leukemia

WTAP is a novel oncogenic protein in acute myeloid leukemia

Small-Molecule Inhibitors of USP1 Target ID1 Degradation in Leukemic Cells

Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhodamine-based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner. A known USP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy.

A-Type Proanthocyanidins Prevent Engraftment Of Primary Acute Myelogenous Leukemia Cells In Mice and Exhibit Potentially Novel Anti-Leukemia Mechanisms

Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. AML originates and is maintained by leukemia stem cells (LSCs). Failure to eliminate LSCs by chemotherapy is likely to result in disease relapse. Therefore, it is a priority to identify new therapies that eliminate blasts while ablating LSCs and preventing a relapse.We have found that a unique class of compounds in cranberries (Vaccinium macrocarponAit.), known as A-type proanthocyanidins (A-PACs), were effective against several leukemia cell lines and primary AML samples in vitro. A-PACs consist of monomeric epicatechin units attached to one another by a carbon-carbon bond and a distinctive ether bond that differentiates these compounds from other proanthocyanidins found in nature. Moreover, A-PACs possess ortho-hydroxyl phenolic groups that have the potential to bind to iron and alter redox status.Preliminary work showed that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), while normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction of A-PACs of up to 7 degree of polymerization was more effective than individual A-PACs. This information prompted us to investigate the in vivo anti-leukemia effects of A-PACs in xenotransplanted mice with primary AML samples, and to further investigate the mechanisms associated with these compounds.Primary AML cells were injected in sub-lethally irradiated NOD/SCID mice. Four weeks after injections, when human leukemia cells have engrafted, intraperitoneal injections of cytarabine (AraC) at 60 mg/kg were given to the mice for 1 week everyday or A-PACs (100 mg/kg dose every 3 days for A-PACs) and vehicle control (1% DMSO in PBS every 3 days) were injected for 2.5 weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, ROS, and survival pathways either by gene expression analysis, flow cytometry or mass spectrometry.Administration of A-PACs to NOD-SCID mice bearing AML tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 16.1% (95% CI: -6.0, 38.37; n=4), whereas the mice treated with the A-PACs and AraC showed a level of engraftment of 4.9% (95% CI: 2, 8; n=5) and 5.8% (95% CI: -1.1, 12.7; n=5), respectively. No significant changes in hemoglobin or weight were found between the different treatment groups. Moreover, qPCR analysis of sensitive leukemia cell lines treated with A-PACs showed changes in gene expression of several iron metabolism genes in sensitive leukemia cell lines (up-regulation of ferritin and transferrin receptors 1 and down-regulation of ferroportin) and several ROS-relevant genes (down-regulation of nuclear factor erythroid-2-related factor 2 and glutamate-cysteine ligase regulatory subunit). Mass spectrometry also confirmed that A-PACs bind iron.The results indicate that A-PACs not only target primary AML cells in vitro but are also effective in vivo. Secondary transplants are also being performed to determine the effects on LSC activity. Some of the anti-leukemia mechanisms under investigation include effects related to iron metabolism, ROS or inhibition of survival pathways. Understanding the unique structure and biological effects of A-PACs may provide novel information about pathways involved in the survival of LSCs and provide crucial information in preparation for clinical trials and/or optimal combination drug therapies. Disclosures:Rivella:Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Activation Of Akt Enhances Ribosomal RNA Synthesis In AML Cells Through a Novel Isoform Of TIF-1A and Inhibition Of Filamin A Cleavage

PI3Kinase/Akt signaling is frequently activated in acute myelogenous leukemia (AML) and inhibitors of this pathway have shown some promise in both pre-clinical and early clinical studies. In particular, much attention has been focused on components of the mTOR complex as downstream effectors of Akt activation that promote cell proliferation and survival in AML and other malignancies. We have discovered a direct effect of activated and phosphorylated Akt in increasing ribosomal RNA (rRNA) synthesis in both leukemic cell lines and primary AML cells by a novel pathway that is independent of mTOR. We have defined TIF-90 as a splice variant of TIF-1A, a molecule that tethers RNA Polymerase I to the ribosomal DNA promoter and is required for rRNA synthesis. TIF-90 is localized in the nucleolus and directly co-locates with Pol I in imaging, pull down, and rDNA promoter occupancy studies. Activation and phosphorylation of Akt correlates with high levels of TIF-90 expression and increased TIF-90 occupancy of the rDNA promoter in primary AML cells, while overexpression of constitutively activated Akt (Akt-Myr) increases rRNA synthesis in a TIF-90-dependent fashion in cell lines. Activation of Akt increases the expression of TIF-90 by inhibiting its ubiquitination and proteasomal degradation by the p53 ubiquitin ligase, Mdm2. Furthermore, activated Akt directly inhibits the cleavage of the actin-binding protein, Filamin A, into a 90 kDa fragment that interacts with TIF-90 and prevents it from occupying the rDNA promoter. Expression of the 90 kDa Filamin A cleavage fragment alone is sufficient to inhibit rRNA synthesis by binding to TIF-90. Thus, inhibition of Filamin A cleavage by Akt is associated with increased rRNA synthesis. The Akt inhibitor AZD8055, but not the mTORC1 inhibitor Rapamycin, promotes the cleavage of filamin A, decreasing both TIF-90 occupancy of the rDNA promoter and rRNA synthesis. Examination of 21 primary AML samples reveals an inverse correlation between the phosphorylation of Akt and the cleavage of Filamin A; conversely, AZD8055 inhibits both Akt phosphorylation and rRNA synthesis in these samples while promoting the cleavage of Filamin A. These data demonstrate a novel pathway by which Akt directly enhances rRNA synthesis. This pathway is active in primary AML cells and in cell lines that have high levels of phosphorylated Akt. Inhibition of rRNA synthesis has been shown to result in nucleolar disruption, cell cycle arrest, and ultimately cell death. Our data support an mTOR-independent effect of Akt activation on cell proliferation that would require direct targeting of PI3Kinase or activated Akt to have a major therapeutic effect in AML. Disclosures:No relevant conflicts of interest to declare.

The pp90rsk-CREB Signaling Pathway Regulates Apoptosis In Acute Myelogenous Leukemia

CREB (cAMP Response-Element Binding Protein) is a nuclear transcription factor critical for hematopoietic cell proliferation, differentiation, and survival. We previously demonstrated that 60% of patients with Acute Myelogenous Leukemia (AML) overexpress CREB in leukemic blasts, and CREB overexpression in these patients was associated with an increased risk of relapse and decreased event-free survival. Previous studies have suggested that CREB may play an important role in the regulation of apoptosis in a wide variety of cancers. Specifically, CREB has been shown to up-regulate members the anti-apoptotic protein family such as Bcl-2, Bcl-XL and Mcl-1, leading to chemotherapy resistance in vitro. CREB-mediated resistance to apoptosis may underlie the increased rate of relapse and poor survival of AML patients with CREB overexpression. Thus, we hypothesized that targeted inhibition of CREB in AML cells would promote AML cell apoptosis. To test this hypothesis, we developed a small-molecule inhibitor of CREB function, XX-650-23. This molecule disrupts the interaction between CREB and its binding partner CBP (CREB-Binding Protein), which is required for full activation of CREB-mediated gene transcription. Treatment of primary AML patient bone marrow samples with XX-650-23 induced apoptosis and cell death at a dose of 2 uM. The degree of apoptosis varied with the expression level of CREB in primary AML cells tested. Higher CREB levels correlated with higher sensitivity to XX-650-23. In non-leukemic primary patient bone marrow samples, CREB levels were very low, and XX-650-23 did not induce apoptosis in these cells. AML cell lines (KG-1 and HL-60) also underwent apoptosis following CREB inhibition, in proportion to CREB expression level. CREB knockdown or overexpression in KG-1 cells decreased and increased susceptibility to apoptosis, respectively. Mechanistically, the onset of apoptosis in AML cells occurred simultaneously with down-regulation of Bcl-2, a validated CREB-regulated gene. Inhibition of Bcl-2 function using the specific Bcl-2 inhibitor ABT-737 (100 nM) induced apoptosis similar to XX-650-23, indicating that Bcl-2 inhibition alone is sufficient to cause apoptosis. Thus, targeted inhibition of CREB results in Bcl-2 downregulation and is sufficient to induce apoptosis in AML cells. Proteomic analysis using Mass Cytometry-Time of Flight (CyTOF) revealed that one compensatory cellular response to CREB inhibition is increased phosphorylation of CREB. This phosphorylation decreased in the presence of BI-D1870, a specific inhibitor of the pp90RSK kinase (RSK), but not by pharmacologic inhibition of the p38 or ERK kinases, using SB202190 or U0126, respectively. We therefore examined the role of pp90RSK in the regulation of apoptosis in AML cells. Pharmacologic inhibition of RSK independently lead to AML cell apoptosis (BI-D1870, IC50=3.3 uM), in part due to blockade of CREB phosphorylation. In summary, our data provide the first evidence that inhibition of CREB, or its chief activator RSK, is sufficient to induce apoptosis in AML cells. Current work focuses on defining CREB target genes mediating XX-650-23 response using chromatin-immunoprecipitation with massively parallel DNA sequencing (ChIP-Seq), and defining the RSK kinome in AML cells using 2-dimensional gel phosphoprotein profiling. These studies will more fully define the role of the RSK-CREB signaling axis in AML proliferation, survival, and apoptosis. Disclosures:No relevant conflicts of interest to declare.

A Phase 1 Study Of TH-302, An Investigational Hypoxia-Targeted Drug, In Patients With Advanced Leukemias

BackgroundTH-302 is a 2-nitroimidazole prodrug of the DNA alkylator bromo-isophosphoramide mustard designed to be selectively activated in hypoxic conditions. Preclinical data in mice with acute lymphoblastic leukemia (ALL) have demonstrated marked expansion of hypoxia in areas of marrow leukemia infiltrates (Benito et al., PLoS One 2011). TH-302 also exhibited specific hypoxia-dependent cytotoxicity when tested against primary ALL and acute myelogenous leukemia (AML) samples in vitro (Benito et al., ASH 2012). Based on these findings, a phase 1 study of TH-302 was designed for advanced leukemias. MethodsEligible patients had ECOG ≤ 3, relapsed/refractory leukemias for which no standard therapy options were available, and acceptable hepato-renal function. A standard 3+3 dose-escalation design was used with 40% dose increments. TH-302 was administered IV over 30-60 min daily, either by 30 min-bolus administration or as a continuous infusion on days 1-5 of a 21-day cycle. The objectives were to determine the maximum tolerated dose (MTD) and pharmacokinetic (PK) profile of TH-302 with these schedules and to assess preliminary clinical activity of TH-302. ResultsA total of 49 patients with previously treated AML (n=39), ALL (n=9) or CML in blast phase (n=1) received TH-302 at bolus doses of 120 (n=4), 170 (n=4), 240 (n=3), 330 (n=3), 460 (n=20), 550 (n=4) mg/m2 or continuous doses of 330 (n=8) or 460 (n=3) mg/m2. Two of 3 evaluable patients treated with bolus TH-302 (550 mg/m2) experienced DLTs of grade 3 esophagitis; bolus administration MTD was established at 460 mg/m2. Two of 3 patients treated with continuous infusion of TH-302 (460 mg/m2) experienced DLTs of grade 3 mucositis or grade 3 hyperbilirubinemia; continuous administration MTD was established at 330 mg/m2. Thirteen patients received greater than 1 cycle. Generally, a significant rapid cytoreduction was evident early in the cycle, but was not maintained prior to initiation of the next cycle. Two AML patients who received 550 mg/m2 bolus TH-302 had complete resolution of leukemia cutis. One AML patient at 550 mg/m2 bolus TH-302 had a complete response with incomplete platelet recovery (CRp), and one AML patient at 440 mg/m2 bolus TH-302 had a CR. ConclusionsIn patients with advanced leukemias, MTDs were established for daily bolus infusion and 5-day continuous infusion of TH-302 at 460 mg/m2 and 330 mg/m2, respectively. Increased incidence of skin and mucosal toxicity were observed at higher dose levels with both administration schedules. Clinical activity of single-agent TH-302 has been noted with a few objective responses, but the majority of cytoreductions were transient. Disclosures:Konopleva:Threshold Pharmaceuticals: Research Funding. Handisides:Threshold Pharmaceuticals: Employment, Equity Ownership. Kroll:Threshold Pharmaceuticals: Employment, Equity Ownership. Kantarjian:Ariad: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; BMS: Research Funding.

DPP4 (CD26) Truncation Of GM-CSF and IL-3 Alters Their Signaling and Functional Activity In Normal and Leukemic Hematopoietic Stem and Progenitor Cells

DPP4 (CD26) is a dipeptidyl peptidase that functions by enzymatically cleaving the penultimate proline, alanine or select other amino acids such as serine of proteins, resulting in functional alterations of the protein. We recently published that many cytokines, chemokines and growth factors have putative DPP4 truncations sites and that DPP4 specifically was able to truncate some colony stimulating factors such as GM-CSF and IL-3 with resultant blunting of their activity. However, the mechanism of action of the truncated factors is still unknown and requires further investigation. The expression, and activity, of DPP4 is relevant in normal and malignant hematopoiesis as we have data showing that CD34+ umbilical cord blood cells (UCB) as well as Acute Myelogenous Leukemia (AML) patient samples express active DPP4. Further, specific inhibition of DPP4 increases homing and engraftment of both human UCB and mouse bone marrow cells after transplantation in mice indicating the therapeutic potential of DPP4 activity altering compounds. Due to its potential importance in disease states, and their subsequent treatment, it is relevant to study how the activity of DPP4 alters the functions of the molecules it cleaves, and subsequently their interactions with each other.DPP4 can cleave the penultimate proline of GM-CSF and IL-3 resulting in truncated forms which have blunted colony stimulating factor activity for hematopoietic progenitor cells (HPC). Since GM-CSF and IL-3 receptors share a common receptor beta chain, we investigated if DPP4 truncation of GM-CSF (TGM) or IL-3 (T3) could inhibit the receptor binding and functional activity of the full length (FL) alternate compound (i.e TGM inhibition of FL3 activity or T3 inhibition of FLGM activity) in the factor dependent TF-1 cell line, UCB cells and in in vivo mouse studies. We determined using TF-1 and UCB that both T3 and TGM bound to the receptors with higher affinity than their FL forms and could blunt the receptor binding of the FLGM and FL3. Additionally, TGM and T3 decreased colony formation induced by either FLGM or FL3 in both TF-1, UCB, and primary AML patient cell samples. Strikingly, this inhibition of colony formation did not require a 1:1 ratio of the full length to truncated forms of these cytokines. Rather, approximately 4-10 fold less truncated molecules could be used to efficiently inhibit the colony formation activity of the full length form, even across molecules. In vivo injection of FL, T, or a mixture of FL/T or T/T factors into DPP4 activity knockout mice followed by colony assays showed the TGM and T3 suppresed the effect of FLGM or FL3 on progenitor cell numbers per femur and diminished cycling of hematopoietic progenitor cells as detected by high specificity tritiated thymidine kill assay.Proteomic analysis of the effects of full length and truncated factors (FLGM, FL3, TGM, T3) were performed with TF-1 cells where we detected differential protein regulation by the full length vs truncated factors. After 24 hour treatment with 10ng/ml of FLGM or TGM, TF-1 cells displayed statistically significant (p < .05) differences in 26 proteins of which 17 were higher in the FL vs the T, and 9 higher in the T vs FL treated groups. These proteins included, but were not limited to, cell cycle proteins such as CDK6, HDAC6, as well as signal transduction proteins and redox control proteins such as STAM1 and Glutaredoxin. Additionally, alterations in protein phosphphorylation were detected for TF-1 cells treated for 15 or 30 min with the full length vs truncated IL-3 and GM-CSF proteins. Interestingly, the protein expression or phosphorylation levels were not always decreased by the truncated protein compared to the full length. In some cases, the truncated molecules induced an increase in the protein expression or phosphorylation.These data suggest interesting roles for full length and truncated GM-CSF and IL-3 in both normal and malignant hematopoiesis. Further investigation into the regulation of DPP4, and the roles that full length and truncated factors play during normal and malignant hematopoiesis, are important and will allow for a better understanding of the signficance of DPP4 activity during steady state, stressed, and disease hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Panobinostat, An Oral Pan-Histone Deacetylase (HDAC) Inhibitor Activates a Microrna Signature That Targets Rad51 To Attenuate Homologous DNA Repair and Sensitize AML Cells To Sapacitabine

Acute myelogenous leukemia (AML) is characterized by multiple genetic and epigenetic abnormalities including a profound dysregulation of microRNA expression. Specific expression signatures of miRNA are associated with disease behavior and clinical outcome.Effective clinical treatment of AML has largely depended on a class of antimetabolites – the nucleoside analogs. Of these, sapacitabine is a nucleoside analog prodrug that is in development for the therapy of AML. It is converted to its active metabolite 2-C-cyano-2-deoxy-1-β(-D-arabino-pentafuranosyl) cytosine (CNDAC), which interferes with DNA synthesis by initially causing a single stranded DNA break that is converted into a double strand break in the subsequent replicative cycle. Such double strand breaks are primarily repaired by the homologous recombination repair (HR) pathway. Consequently, efficient HR may offer a potential resistance mechanism to therapy with sapacitabine.Panobinostat and vorinostat are pan HDAC inhibitors (HDACi) that modulate gene expression, induce apoptosis, and are clinically active against specific leukemias and lymphomas. In pilot experiments, we identified that exposure of AML cell lines to a combination of panobinostat or vorinostat and sapacitabine had a greater than additive effect on cell viability. Loss of cell survival was preceded by a rapid and marked decline in the levels of Rad51, a protein that is critical in repairing CNDAC-induced damage via the HR repair pathway, both in AML cell lines and primary tumor cells that were exposed to either panobinostat or vorinostat. Therefore, we hypothesized that exposure of AML cells to HDACi would activate the expression of a set microRNAs, that would target Rad51. Loss of Rad51 protein, would in turn impede the successful completion of HR rendering the cells incapable of repairing the damage caused by CNDAC and thus sensitizing them to the nucleoside analog.Our preliminary data using microRNA arrays indicated that exposure of AML cells to panobinostat resulted in the induction of nine microRNA that putatively target Rad51. We then ectopically expressed three of the nine Rad51- directed miRNA and found that miR-182 efficiently targeted the Rad51 protein. Using real time-PCR we found that the levels of miR-182 were significantly lower in primary AML cells when compared to hematopoietic cells from normal donors. Using chromatin immunoprecipitation (ChIP) assays we identified that HDAC1 and HDAC2 were recruited to the miR182 promoter in AML cell lines as well as in primary AML cells, which was probably linked to loss of gene expression. Correspondingly, HDAC inhibition led to the accumulation of activating chromatin modifications at the miR-182 promoter, which was followed by the induction of miR-182 levels in both AML cells lines and primary AML cells. To determine whether HDAC inhibition targets Rad51 via miR-182, we expressed antagomiRs that interfere with the binding of miR-182 to Rad51, and found that the levels of Rad51 were preserved in cells expressing anti-miR182 before being exposed to panobinostat. Finally, we identified that exposure to panobinostat suppressed ongoing HR as measured by the absence of Rad51 foci in cells exposed to this agent, whereas cells exposed to CNDAC alone exhibited robust Rad51 foci formation, indicating an active HR pathway.In conclusion, HDAC inhibition results in the induction of miR-182 which targets Rad51 and attenuates HR to sensitize cells to sapacitabine in AML. This therapeutic strategy may be effective in circumventing potential resistance mechanisms to nucleoside analog therapy in AML. Disclosures:Ewald:Novartis: Employment.

Clinical “MUTATOME” Of Myelodysplastic Syndrome; Comparison To Primary Acute Myelogenous Leukemia

Chromosomal aberrations and somatic mutations constitute key elements of the pathogenesis of myelodysplastic syndromes (MDS), a clonal hematologic malignancy characterized by cytopenias, a dysplastic bone marrow and propensity to clonal evolution. Next generation sequencing (NGS) enables definition of somatic mutational patterns and clonal architecture as a discovery platform, and for clinical applications.We systematically applied NGS to 707 cases of MDS and MDS-related disorders. 205 cases (low-risk MDS: N=78, high-risk MDS: N=42, MDS/MPN: N=48 and sAML: N=37) were tested by whole exome sequencing (WES). For validation in an additional 502 patients (low-risk MDS: N=192, high-risk MDS: N=104, MDS/MPN: N=111 and sAML: N=95), targeted deep NGS was applied for 60 index genes which were most commonly affected in the cohort analyzed by WES. For NGS data analysis a statistical pipeline was developed to focus on: i) identification of the most relevant somatic mutations, and ii) minimization of false positive results. We studied serial samples from 21 exemplary informative patients. We also compared somatic mutational patterns to those seen in primary AML TCGA cohort (N=201). Given the size of the cohort, there was, for example, a 87% chance of seeing mutations at a frequency of 1% and a 98% of seeing those with a frequency of 2%. While focusing on the most common events, we observed 1117 somatic mutations in 199 genes. The 88 genes mutated mutated in >1% of cases with MDS carried 388 mutations in MDS+sAML (2.5/case), 128 in MDS/MPN (2.7/case) and 398 in pAML (2.0/case). The average number of mutations per case increased during progression (2.2 in lower-risk, 2.8 in higher-risk MDS, 3.4 in sAML). In MDS, the 30 most frequently affected genes were present at least once in 70% of patients. The 30 most frequently mutated genes in MDS/MPN were mutated in 82% of patients. Individual mutations were also sub-grouped according to their function. When we compared three MDS subcategories (lower-risk, higher-risk MDS and sAML) in a cross-sectional view, RTK family, RAS family, IDH family and cohesin family mutations were more frequently detected in the sAML group than in the MDS group. In contrast, the frequency of the DNMT family, TET2 and ASXL family gene mutations did not increase in frequency in the sAML cohort. In addition to better definition of mutational patterns of known genes, we have also defined new mutations, including in the RNA helicase family and the BRCC3pathway.Clonal architecture analysis indicates that mutations of TET2, DNMT3A, ASXL1, and U2AF1 most likely represent ancestral/originator events, while those of the IDH family, RTK family and cohesin family are typical secondary events. Establishment of mutational patterns may improve the precision of morphologically-based diagnosis. The comparison between MDS-related diseases (MDS+sAML) and pAML revealed a notably different mutational pattern suggestive of a distinct molecular derivation of these two disease groups. While RTK, IDH family and NPM1 mutations were more frequently observed in the pAML cohort, mutations of SF3B1 and SRSF2, were more common in MDS+sAML. With regard to the connections between individual mutation combinations, RTK mutations were strongly associated with DNMT, but not with RAS family mutations in the pAML cohort, while the mutual association between TET2 and PRC2 family, cohesin family and RUNX1were encountered in the MDS+sAML cohort.Individual mutations may have prognostic significance, including having an impact on survival, either within the entire cohort or within specific subgroups. In the combined MDS cohort, TP53 family mutations were associated with a poor prognosis (HR; 3.65, 95%CI; 1.90-7.01, P<.0001) by univariate analysis. Similar results were found for mutations in TCF4(HR; 7.98, 95%CI; 1.58-10.1, P<.0007). Such an individual approach does not allow for assessment of the impact of less common mutational events.In conclusion, our study continues to indicate the power of NGS in the molecular analysis of MDS. MDS and related disorders show a great deal of pathogenetic molecular overlap, consistent with their morphologic and clinical pictures, but also distinct molecular differences in mutational patterns. Some of the specific mutations are pathognomonic for specific subtypes while some may convey a prognostic rather than discriminatory value. Disclosures:Makishima:Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding. Polprasert:MDS foundation: Research Funding.

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

Differences in proliferative capacity of primary human acute myelogenous leukaemia cells are associated with altered gene expression profiles and can be used for subclassification of patients

Proliferative capacity of acute myelogenous leukaemia (AML) blasts is important for leukaemogenesis, and we have investigated whether proliferative capacity of primary human AML cells could be used for subclassification of patients. In vitro proliferative capacity of AML cells derived from two independent groups was investigated. Cells were cultured under highly standardized conditions and proliferation assayed by (3) H-thymidine incorporation after seven days culture. Patients were subclassified by clustering models, and gene expression profile was examined by microarray analyses. Based on proliferative capacity of the AML cells, three different patient clusters were identified: (i) autocrine proliferation that was increased by exogenous cytokines; (ii) detectable proliferation only in presence of exogenous cytokines; and (iii) low or undetectable proliferation even in presence of exogenous cytokines. Patients with highest proliferative capacity cells had no favourable prognostic impact by NPM-1 mutation. Analysis of gene expression profiles showed that the most proliferative cells generally had altered expression of genes involved in regulation of transcription/RNA functions, whereas patients with high proliferative capacity and internal tandem duplications (ITDs) in the FLT3 cytokine receptor gene had altered expression of several molecules involved in cytoplasmic signal transduction. In vitro proliferative capacity of primary human AML cells was considerably variable between patients and could be used to identify biologically distinct patient subsets.

Role of cysteine 288 in nucleophosmin cytoplasmic mutations: sensitization to toxicity induced by arsenic trioxide and bortezomib

Mutations in exon 12 of the nucleophosmin (NPM1) gene (NPMc+ (NPM1 COOH terminal mutations)) define a distinct subset of acute myelogenous leukemias (AMLs), in which the NPMc+ protein localizes aberrantly to the leukemic cell cytoplasm. We have found that introduction of the most common NPMc+ variant into K562 and 32D cells sensitizes these cells to apoptosis induced by drugs such as bortezomib and arsenic trioxide (ATO) that induce reactive oxygen species (ROS) formation, and that cytotoxicity is prevented in the presence of N-acetyl-L-cysteine (NAC), an ROS scavenger. The substitution of tryptophan 288 (W288) by cysteine occurs in the great majority of NPM1c+ mutations. Mutagenesis of cysteine 288 to alanine re-localizes NPMc+ from the cytoplasm to the nucleolus and attenuates the sensitivity of cells expressing this mutation to bortezomib and ATO. Primary AML cells expressing NPMc+ are also significantly more sensitive than other AML cells to apoptosis induced by both drugs at pharmacologically achievable doses. We conclude that the presence of a cysteine moiety at position 288 results in the cytoplasmic localization of NPM1c+ and the increased sensitivity to bortezomib and ATO. These data suggest that bortezomib and ATO may have increased therapeutic efficacy in NPM1c+ leukemias.

The Novel Chk1 Inhibitor MK-8776 Sensitizes Human Leukemia Cells to HDAC Inhibitors by Targeting the Intra-S Checkpoint and DNA Replication and Repair

Interactions between the novel Chk1 inhibitor MK-8776 and the histone deacetylase (HDAC) inhibitor (HDACI) vorinostat were examined in human leukemia cells harboring wild-type (wt) or deficient p53. MK-8776 synergistically potentiated vorinostat-mediated apoptosis in various p53-wt or -deficient leukemia cell lines, whereas p53 knockdown by short hairpin RNA (shRNA) sensitized p53-wt cells to lethality of this regimen. Leukemia cell lines carrying FLT3-ITD were also sensitive to the MK-8776/vorinostat regimen. Synergistic interactions were associated with inhibition of Chk1 activity, interference with the intra-S-phase checkpoint, disruption of DNA replication, and downregulation of proteins involved in DNA replication (e.g., Cdt1) and repair (e.g., CtIP and BRCA1), resulting in sharp increases in DNA damage, reflected by enhanced γ-H2A.X formation, and apoptosis. Moreover, leukemia cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared with their wt counterparts and displayed downregulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Finally, the MK-8776/vorinostat regimen was active in primary acute myelogenous leukemia (AML) blasts, particularly against the CD34(+)/CD38(-)/CD123(+) population enriched for leukemia-initiating cells. In contrast, identical regimens were relatively sparing toward normal cord blood CD34(+) cells. Together, these findings indicate that the novel Chk1 inhibitor MK-8776 markedly potentiates HDACI lethality in leukemia cells displaying various genetic backgrounds through mechanisms involving disruption of the intra-S checkpoint, DNA replication, and DNA repair. They also argue that leukemic cells, including those bearing oncogenic mutations associated with poor prognosis, for example, p53 deletion/mutation or FLT3-ITD, may also be susceptible to this strategy.

Overexpression of hCLP46 enhances Notch activation and regulates cell proliferation in a cell type‐dependent manner

Human CAP10-like protein 46 kDa (hCLP46), also known as Poglut1, has been shown to be an essential regulator of Notch signalling. hCLP46 is overexpressed in primary acute myelogenous leukaemia, T-acute lymphoblastic leukaemia samples and other leukaemia cell lines. However, effects of hCLP46 overexpression, up to now, have remained unknown. In this study, we established stable 293TRex cell lines inducibly overexpressing hCLP46, and knocked down hCLP6 with a specific small interfering RNA to explore function of the protein in Notch signalling and cell proliferation. hCLP46 overexpression enhanced Notch1 activation in 293Trex cells in a ligand-dependent manner, with increased Notch signalling enhancing Hes1 expression. We further verified that overexpression of hCLP46 inhibited proliferation of 293TRexs and was correlated with increases in cyclin dependent kinase inhibitors p21 and p27, whereas reduced hCLP46 expression moderately increased cell proliferation. In addition, p21 and p27 protein levels were higher when Notch signalling was activated by EDTA treatment. Taken together, hCLP46 enhanced Notch activation and inhibited 293TRex cell proliferation through CDKI signalling.

Abstract 3396: Improving the anti-AML efficacy of liposomal ceramide by targeting sphingolipid metabolism.

Abstract Novel approaches to enhancing the delivery, uptake, and therapeutic effect of sphingolipids are of interest for the treatment of neoplasms. We attempt to promote cellular ceramide accumulation in murine and human models of acute myelogenous leukemia (AML) by modifying ceramide metabolism with either the sphingosine kinase1 inhibitor safingol (Lip-Saf), or the acid ceramidase inhibitor LCL204, preventing ceramide glycosylation by inhibition of p-glycoprotein (P-GP) activity with tamoxifen (Lip-Tam), and supplementing cells with exogenous C6 ceramide (Lip-C6) via liposomal delivery. Ceramide is widely recognized as an apoptotic signal and blocking its catabolism to sphingosisne-1-phosphate has been shown to induce autophagy. Safingol and LCL204 both modify ceramide catabolism. Supplementation of tamoxifen has been shown to promote apoptosis via ceramide accumulation by inhibiting the activity of P-GP at the Golgi membrane, which can participate in ceramide glycosylation. C1498, HL-60, HL-60/VCR, and KG-1 leukemia cell line viability was quantified following treatment with Lip-C6, Lip-Saf, or a 2:1 combination to measure synergy. Likewise, cell line viability was evaluated using Lip-Tam and Lip-C6, independently and in a 1:1 combination to study synergy. C1498 viability was also quantified with increasing concentrations of LCL204. CalcuSyn software was used to analyze dose-response and synergistic therapeutic efficacy. Colony-forming capacity was assessed using a poor-prognosis AML patient sample treated with Lip-C6, Lip-Saf, or the combination of both. Autophagy and apoptosis were further evaluated using flow cytometry with primary AML samples and leukemia cell lines. A reduction in IC50 of the drugs used in combination was noted in several cell lines. Dramatic IC50 shifts were observed in KG-1, HL-60, and the vincristine-resistant HL-60/VCR cell lines after exposure to a combination of Lip-C6 and Lip-Saf in a 2:1 ratio. Flow cytometry confirmed Lip-Saf enhances autophagy, whereas Lip-C6 exerts an effect primarily through apoptosis in C1498 and primary AML samples. Therefore, the observed cell death synergism is likely due to induction of both autophagy and apoptosis. Additionally, it was shown that the anti-AML efficacy of Lip-Tam was dramatically enhanced in combination with Lip-C6, demonstrating that inhibition of ceramide glycosylation is also a valid combinatorial therapeutic approach. Lastly, inhibition of acid ceramidase via LCL204 demonstrated efficacy as a cytotoxic agent both in vitro and in vivo using C1498 cells alone or engrafted into C57BL/6J mice. These studies verify inhibition of ceramide metabolism as an effective strategy to enhance the therapeutic efficacy of ceramide in the treatment of AML. Citation Format: Timothy J. Brown, Aileen M. Garcia, Su-Fern Tan, Lindsey N. Kissinger, David J. Feith, Brian M. Barth, David F. Claxton. Improving the anti-AML efficacy of liposomal ceramide by targeting sphingolipid metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3396. doi:10.1158/1538-7445.AM2013-3396

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34(+) cells, especially the LSC-enriched CD34(+)CD38(-) subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34(+) cells but not those from normal CD34(+) cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide-down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.

Dual Inhibition of Bcl-2 and Bcl-xL Strikingly Enhances PI3K Inhibition-Induced Apoptosis in Human Myeloid Leukemia Cells through a GSK3- and Bim-Dependent Mechanism

Effects of concomitant inhibition of the PI3K/AKT/mTOR pathway and Bcl-2/Bcl-xL (BCL2L1) were examined in human myeloid leukemia cells. Tetracycline-inducible Bcl-2 and Bcl-xL dual knockdown sharply increased PI3K/AKT/mTOR inhibitor lethality. Conversely, inducible knockdown or dominant-negative AKT increased, whereas constitutively active AKT reduced lethality of the Bcl-2/Bcl-xL inhibitor ABT-737. Furthermore, PI3K/mTOR inhibitors (e.g., BEZ235 and PI-103) synergistically increased ABT-737-mediated cell death in multiple leukemia cell lines and reduced colony formation in leukemic, but not normal, CD34+ cells. Notably, increased lethality was observed in four of six primary acute myelogenous leukemia (AML) specimens. Responding, but not nonresponding, samples exhibited basal AKT phosphorylation. PI3K/mTOR inhibitors markedly downregulated Mcl-1 but increased Bim binding to Bcl-2/Bcl-xL; the latter effect was abrogated by ABT-737. Combined treatment also markedly diminished Bax/Bak binding to Mcl-1, Bcl-2, or Bcl-xL. Bax, Bak, or Bim (BCL2L11) knockdown or Mcl-1 overexpression significantly diminished regimen-induced apoptosis. Interestingly, pharmacologic inhibition or short hairpin RNA knockdown of GSK3α/β significantly attenuated Mcl-1 downregulation and decreased apoptosis. In a systemic AML xenograft model, dual tetracycline-inducible knockdown of Bcl-2/Bcl-xL sharply increased BEZ235 antileukemic effects. In a subcutaneous xenograft model, BEZ235 and ABT-737 coadministration significantly diminished tumor growth, downregulated Mcl-1, activated caspases, and prolonged survival. Together, these findings suggest that antileukemic synergism between PI3K/AKT/mTOR inhibitors and BH3 mimetics involves multiple mechanisms, including Mcl-1 downregulation, release of Bim from Bcl-2/Bcl-xL as well as Bak and Bax from Mcl-1/Bcl-2/Bcl-xL, and GSK3α/β, culminating in Bax/Bak activation and apoptosis. They also argue that combining PI3K/AKT/mTOR inhibitors with BH3 mimetics warrants attention in AML, particularly in the setting of basal AKT activation and/or addiction.

MiR-24 Promotes the Survival of Hematopoietic Cells

The microRNA, miR-24, inhibits B cell development and promotes myeloid development of hematopoietic progenitors. Differential regulation of cell survival in myeloid and lymphoid cells by miR-24 may explain how miR-24′s affects hematopoietic progenitors. MiR-24 is reported to regulate apoptosis, either positively or negatively depending on cell context. However, no role for miR-24 in regulating cell death has been previously described in blood cells. To examine miR-24′s effect on survival, we expressed miR-24 via retrovirus in hematopoietic cells and induced cell death with cytokine or serum withdrawal. We observed that miR-24 enhanced survival of myeloid and B cell lines as well as primary hematopoietic cells. Additionally, antagonizing miR-24 with shRNA in hematopoietic cells made them more sensitive to apoptotic stimuli, suggesting miR-24 functions normally to promote blood cell survival. Since we did not observe preferential protection of myeloid over B cells, miR-24′s pro-survival effect does not explain its promotion of myelopoiesis. Moreover, expression of pro-survival protein, Bcl-xL, did not mimic miR-24′s impact on cellular differentiation, further supporting this conclusion. Our results indicate that miR-24 is a critical regulator of hematopoietic cell survival. This observation has implications for leukemogenesis. Several miRNAs that regulate apoptosis have been shown to function as either tumor suppressors or oncogenes during leukemogenesis. MiR-24 is expressed highly in primary acute myelogenous leukemia, suggesting that its pro-survival activity could contribute to the transformation of hematopoietic cells.

RNA Trafficking by Acute Myelogenous Leukemia Exosomes

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML.

T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of AML blasts by a novel CD33/CD3-bispecific BiTE antibody construct

Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N=23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1 ng/ml (1.8 pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML.