Acute Myelogenous Leukemia Patient Samples Research Articles

Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia.

Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a molecular weight exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs. We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33+ human leukemia cell lines, xenograft models, and AML patient samples. Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33+ AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T cells. Activation and proliferation of T cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor density and cytogenetic risk. Tumor growth delay and inhibition were observed in both prophylactic and established HL-60 xenograft models in immunodeficient mice. Our data show high efficacy of CD33/CD3 TandAbs in various preclinical models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML. Clin Cancer Res; 22(23); 5829-38. ©2016 AACR.

Abstract 3499: Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia

Abstract Introduction Standard treatment for acute myelogenous leukemia (AML) has not changed over the past few decades and relies primarily on the traditional “7 + 3″ regimen of daunorubicin, administered over 3 days and Cytarabine, administered over 7 days. This chemo-intensive regimen is poorly tolerated by patients and is characterized by a high rate of relapse and treatment failure. Triptolide is a diterpenoid tri-epoxide compound isolated from the Chinese herb Tripterygium wilfordii. The water-soluble pro-drug of triptolide, Minnelide has been shown by our group to be highly effective in a number of pre-clinical models of solid tumors and is currently undergoing Phase I Clinical trial for gastrointestinal tumors. Interim analysis of the Phase I data has shown that the equivalent mouse dose of 0.2 mg/kg/day is well tolerated by patients with no reported dose limiting toxicity. Methods Primary AML apheresis samples from patients with acute myeloid leukemia and multiple AML cell Lines (THP1, KG1, Kasumi1, HL-60) were treated with Triptolide at doses from of 2.5 nM to 50nM and cell viability was measured using a formazan based colorimetric assay. Apoptosis was measured using Annexin V via flowcytometry and colony forming ability of AML cell lines was measured using a methylcellulose based assay. To generate an engraftment model of AML, NOD.Rag1-/-;γcnull (NRG) animals expressing human interleukin-3 (IL-3) and human GM-CSF (NRGS) were injected luciferase expressing THP1 cells after sublethal irradiation of 250 cGy. These animals were then serially evaluated using In Vivo Imaging System (IVIS) and leukemic burden was calculated the total bioluminescent signal after intra-periotneal injection of luciferin. Treatment with vehicle or Minnelide at a dose of 0.1mg/kg/day and 0.15 mg/kg/day was started on the 10th day after confirming engraftment using IVIS. Results Triptolide at a dose range of 2.5 nM to 50 nM produced a dose and time dependent killing of leukemic cells in both cell lines and primary AML patient samples. The IC-50 for THP1, KG1, Kasumi1 and HL-60 cell lines with triptolide treatment at 48 hours were 5 ± 0.8 nM, 8 ± 0.7 nM, 7.2 ± 0.6 nM, 10 nM ± 2 nM respectively. Treatment with triptolide at a dose of 2.5 nM induced cell death and apoptosis as measured by Annexin V posiitvity in primary AML apheresis samples. Colony formation by THP1 cells and KG1 cells was completely abrogated by treatment with Triptolide at 2.5 nM and 25 nM respectively. Vehicle treated mice showed a rapid progression of disease burden when compared to Minnelide treated mice. At a dose of 0.1 mg/kg/day and 0.15 mg/kg/day, these mice had no appreciable increase in leukemic burden over normal mice when assessed by IVIS. Conclusion We show that Minnelide induces cell death at therapeutically relevant concentrations in vitro and decreased leukemic burden in a murine model of AML. Minnelide may emerge as a novel therapeutic strategy in treating patients with AML. Citation Format: Bhuwan Giri, Sulagna Banerjee, John George, Shrey Modi, Vineet Kumar Gupta, Mahendra K. Singh, Vikas Dudeja, Ashok K. Saluja. Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3499.

BCOR regulates myeloid cell proliferation and differentiation.

BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes.

Abstract 5416: Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous leukemia

Abstract The PIM family of serine-threonine protein kinases (PIM1, PIM2 and PIM3) was initially identified as preferential integration sites of the Moloney murine leukemia virus in Eμ-myc mice, resulting in perinatal lymphomagenesis. Molecular characterization has revealed that PIM kinases drive cell proliferation and survival in a number of hematological malignancies beyond lymphomas by mediating responses to cytokines, growth factors and cellular stress. Overexpression of various PIM kinase family members in these malignancies has been associated with poor overall survival and with resistance to chemotherapeutic agents. Therefore, development of a pan-PIM inhibitor may be useful in the treatment of hematological malignancies, both as a single agent and in combination with chemotherapy or targeted agents. The in vitro and in vivo activity of INCB053914, a pan-PIM kinase inhibitor, was determined in a panel of acute myelogenous leukemia (AML) cell lines. Greater than half of all AML cell lines tested were sensitive to single agent INCB053914, with anti-proliferative IC50 potencies <100 nM. Consistent with recent findings, the majority of these sensitive cell lines expressed higher levels of CD25, which is co-regulated with PIM through the JAK/STAT signaling pathway and which may serve as a biomarker for PIM sensitivity in AML. INCB053914 suppressed the phosphorylation of multiple PIM kinase substrates and, in a compensatory manner, increased PIM kinase expression in both cell lines and primary AML patient samples. The potency of INCB053914 to suppress BAD phosphorylation in vivo in MOLM16 tumors is consistent with its potency in blocking BAD phosphorylation in MOLM16 cells spiked into human whole blood in vitro. Dose dependent tumor growth inhibition (TGI) was seen in mice bearing MOLM16 tumors, with maximal TGI achieved with 12-20 hours of MOLM16 WB IC50 coverage. Synergistic activity was seen in vivo in the KG1 model when INCB053914 was administered with cytarabine, a standard of care therapy for AML. Further studies were undertaken to assess the potential for combined PIM inhibition with other targeted agents. Because the PIM kinases have been shown to regulate c-myc expression levels, agents whose activity has been shown to be influenced by c-myc levels or to regulate c-myc expression were examined. The combination of INCB53914 with the pan-BET inhibitor INCB54329 in MOLM16 xenografts synergistically decreased pBAD and c-myc levels in tumors, resulting in enhanced efficacy. Synergy was also seen when INCB53914 was combined with the pan-FGFR inhibitor INCB54828 in KG1 xenografts harboring the FOP-FGFR1 translocation, suggesting that this combination might be useful in this rare patient population as well as other diseases driven by oncogenic FGFR activation. Taken together, these data support the utility of PIM inhibition in AML patients as monotherapy and in combination with other targeted agents. Citation Format: Holly Koblish, Niu Shin, Leslie Hall, Sybil O'Connor, Qian Wang, Kathy Wang, Lynn Leffet, Maryanne Covington, Krista Burke, Jason Boer, Kevin Bowman, Ke Zhang, Hao Feng, Chu-Biao Xue, Yun-Long Li, Wenqing Yao, Reid Huber, Kris Vaddi, Peggy Scherle. Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5416. doi:10.1158/1538-7445.AM2015-5416

Tetraspanin 3 Is Required for the Development and Propagation of Acute Myelogenous Leukemia

Acute Myelogenous Leukemia (AML) is an aggressive cancer that strikes both adults and children and is frequently resistant to therapy. Thus, identifying signals needed for AML propagation is a critical step toward developing new approaches for treating this disease. Here, we show that Tetraspanin 3 is a target of the RNA binding protein Musashi 2, which plays a key role in AML. We generated Tspan3 knockout mice that were born without overt defects. However, Tspan3 deletion impaired leukemia stem cell self-renewal and disease propagation and markedly improved survival in mouse models of AML. Additionally, Tspan3 inhibition blocked growth of AML patient samples, suggesting that Tspan3 is also important in human disease. As part of themechanism, we show that Tspan3 deficiency disabled responses to CXCL12/SDF-1 and led to defects in AML localization within the niche. These identify Tspan3 as an important regulator of aggressive leukemias and highlight a role for Tspan3 in oncogenesis.

Tyrosine kinase inhibition in leukemia induces an altered metabolic state sensitive to mitochondrial perturbations.

Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL(+) leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph(+) leukemia and AML upon TK inhibition. Ph(+) cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined. We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL(+) leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo. TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL(+) and FLT3(ITD) leukemias.

AG-120, an Oral, Selective, First-in-Class, Potent Inhibitor of Mutant IDH1, Reduces Intracellular 2HG and Induces Cellular Differentiation in TF-1 R132H Cells and Primary Human IDH1 Mutant AML Patient Samples Treated Ex Vivo

▪Point mutations in isocitrate dehydrogenase (IDH) define distinct subsets of acute myelogenous leukemia (AML). IDH is a metabolic enzyme that interconverts isocitrate and α-ketoglutarate (α-KG), but cancer-associated point mutations in IDH1 and IDH2 confer a neomorphic activity that allows reduction of α-KG to the oncometabolite R-2-hydroxyglutarate (2-HG). High levels of 2-HG have been shown to inhibit α-KG-dependent dioxygenases including histone and DNA demethylases, which play a key role in regulating the epigenetic state of cells, but the relationship between 2-HG and oncogenesis is not completely understood. Consistent with 2-HG promoting cancer via an effect on chromatin structure, patients harboring IDH mutations display a CpG island methylator phenotype (CIMP) and several studies have shown that overexpression of IDH mutant enzymes can induce histone and DNA hypermethylation as well as block cellular differentiation. In addition, mice engineered to express IDH1-R132H in hematopoietic tissue have increased early hematopoietic progenitors, splenomegaly, anemia, hypermethylated histones and altered DNA methylation patterns similar to those found in AML patients harboring IDH1/2 mutations.[i] Taken together, these data suggest that cancer-associated IDH mutations may induce a block in cellular differentiation to promote tumorigenesis.To investigate whether selective pharmacological inhibition of the mutant IDH1 enzyme could provide an effective way to lower intracellular 2-HG levels and restore normal differentiation, we treated TF-1 cells or primary human AML patient samples expressing mutant IDH1 with AG-120, an oral, selective, first-in-class, potent IDH1 mutant inhibitor currently in phase I clinical trials. Treatment with AG-120 decreased intracellular 2-HG levels, inhibited growth factor independent proliferation and restored erythropoietin (EPO)-induced differentiation in TF-1 IDH1-R132H cells. Similarly, pharmacological inhibition of mutant IDH1 enzyme with AG-120 in primary human blast cells cultured ex vivo provided an effective way to lower intracellular 2-HG levels and induced myeloid differentiation. Taken together, these data demonstrate that AG-120 is effective at lowering 2-HG levels and restoring cellular differentiation, and support further clinical development of this compound. [Display omitted] PB = peripheral blood, BM = bone marrow [Display omitted] or following 6 days of treatment with control (DMSO) or AG-120 (0.5, 1.0, and 5.0 μM) [Display omitted] [i] M. Sasaki et al., IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics. Nature 488(7413):656-9, 2012. DisclosuresHansen:Agios Pharmaceuticals: Employment, Stockholder Other. Quivoron:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. Straley:Agios Pharmaceuticals: Employment, Stockholder Other. Lemieux:Agios Pharmaceuticals: Employment, Stockholder Other, US20130190249 (pending) Patents & Royalties. Popovici-Muller:Agios Pharmaceuticals: Employment, Stockholder Other. Fathi:Agios Pharmaceuticals: Advisory board participation Other. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. David:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la Recherche contre le Cancer (ARC): Grant, Grant Other; Association Laurette Fugain: Grant, Grant Other; AGIOS: Grant Other. Bernard:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Ligue Nationale contre le cancer (LNCC): Grant, Grant Other; AGIOS: Grant Other. Dorsch:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Su:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. de Botton:AGIOS: Grant Other. Penard-Lacronique:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. Yen:Agios Pharmaceuticals: Employment, Stockholder Other.

Ablation of Autophagy-Related E1 Ligase Atg7 Primes Acute Myelogenous Leukemia Cells to Apoptosis, Sensitizes to Chemotherapy and Overcomes Stroma Mediated Protection

Background: Except for the 'good risk’ groups identified by current cytogenetic and molecular criteria, patients with acute myelogenous leukemia (AML) largely relapse after attaining initial remissions, and outcomes of patients with early relapse is uniformly poor, highlighting the need for strategies to overcome resistance to agents used in frontline therapy. Autophagy is an ancestral adaptive mechanism to stress and can be triggered by exposure to chemotherapy. Autophagy is also believed to play an important role in tumor protection by the microenvironment. Autophagy inhibition increases chemosensitivity in several experimental models of solid tumors. The role of autophagy is not well studied in AML, but the hypoxic bone marrow niche is expected to induce autophagy in AML cells and render them chemo-resistant. Our reverse phase protein array (RPPA) analysis of over 500 newly diagnosed AML patient samples indicated that abnormal expression of several autophagy proteins in leukemic blasts (LKB1, BECLIN 1 and ATG7) is associated with poor prognosis in AML (Borthakur, G et al. ASH 2011# 2513), suggesting a therapeutic role for autophagy suppression. Atg7 is a key molecule in autophagy vesicle elongation with a role in two essential ubiquitin-like reactions (LC3 lipidation and Atg 5/12 conjugation), and its knockdown is expected to inhibit autophagy globally.Aim: To determine the effects of autophagy inhibition on chemosensitivity of AML cells and on stroma induced resistance.Results: We semi-quantitatively assessed ATG7 expression in AML cell lines (THP1, OCI-AML2 and 3, HL-60, MOLT-4, MOLM 13 and 16) by Western blot. HL-60 and MOLM13 cells had the lowest expression of Atg7 and were most sensitive to treatment with ara-C and idarubicin while OCI-AML3 and THP-1 had highest levels of Atg7 and were most resistant to treatment (p= .001-.004). In addition, high expression of Atg7 is associated with high levels of anti-apoptotic Mcl-1 in these lines.As available autophagy inhibitors are non-specific, we used lentiviral shRNA to knock down Atg7 and study the role of autophagy. Atg7 knockdown cells (ATG7-KD) were treated with ara-C and idarubicin for 48-96 hours. At all time points, apoptosis was significantly higher in ATG7-KD cells compared to cells transduced with a non-silencing scrambled control (ATG7-Scr) at 96 hrs: 39.6±2.4 vs 22.4±1.3, p=.002 for ara-C 2 µM and 47.9±2.6 vs 34.7±2.3, p=.0004 for idarubicin. We co-cultured OCI-AML3 (ATG7-KD and -Scr) cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow micro-environment. Sensitivity to ara-C and idarubicin was reduced by co-culture for both cell types, but ATG7-KD cells remained more sensitive than were ATG7-Scr cells.Western blot analysis confirmed increased p62 and decreased LC3 lipidation in ATG7-KD cells (LC3 II/I ratio= 0.15 vs 0.7 in ATG7-Scr), indicating a block in autophagy, and increased caspase 3 cleavage, indicating apoptosis. The higher expression of pro-apoptotic NOXA and BAX, and lower expression of anti-apoptotic MCL-1 and BCL-2, in ATG7-KD cells compare to ATG7-Scr indicating mitochondrial pathway apoptosis. Gene expression profiling on Illumina HT12 arrays was used to study OCI-AML3 ATG7-KD and -Scr cells at baseline and after 24 and 48 hr of araC 0.5 µM. Gene set enrichment analysis (GSEA) showed significantly lower baseline expression of interferon response genes and JAK-STAT pathway genes in OCI-AML3 ATG7-KD cells. Changes with araC treatment were largely similar, but the response to araC was different between ATG7-KD and –Scr cells for a small number of genes. In-vivo chemo-sensitivity experiments are in progress.Conclusion: Autophagy inhibition by genetic silencing of ATG7 increases chemosensitivity of AML cells, even in the presence of bone marrow stromal cells. ATG7-KD cells appear to be more primed for apoptosis compared to their controls. Concomitant inhibition of ATG7, a potentially drugable E1 ligase, appears to be a valid strategy to enhance sensitivity to front-line treatment agents in AML. DisclosuresNo relevant conflicts of interest to declare.

LEO1 Is Regulated by PRL-3 and Mediates Its Oncogenic Properties in Acute Myelogenous Leukemia

PRL-3, an oncogenic dual-specificity phosphatase, is overexpressed in 50% of acute myelogenous leukemia (AML) and associated with poor survival. We found that stable expression of PRL-3 confers cytokine independence and growth advantage of AML cells. However, how PRL-3 mediates these functions in AML is not known. To comprehensively screen for PRL3-regulated proteins in AML, we performed SILAC-based quantitative proteomics analysis and discovered 398 significantly perturbed proteins after PRL-3 overexpression. We show that Leo1, a component of RNA polymerase II-associated factor (PAF) complex, is a novel and important mediator of PRL-3 oncogenic activities in AML. We described a novel mechanism where elevated PRL-3 protein increases JMJD2C histone demethylase occupancy on Leo1 promoter, thereby reducing the H3K9me3 repressive signals and promoting Leo1 gene expression. Furthermore, PRL-3 and Leo1 levels were positively associated in AML patient samples (N=24; P<0.01). On the other hand, inhibition of Leo1 reverses PRL-3 oncogenic phenotypes in AML. Loss of Leo1 leads to destabilization of the PAF complex and downregulation of SOX2 and SOX4, potent oncogenes in myeloid transformation. In conclusion, we identify an important and novel mechanism by which PRL-3 mediates its oncogenic function in AML.

PRMT4 Blocks Myeloid Differentiation by Assembling a Methyl-RUNX1-Dependent Repressor Complex

Defining the role of epigenetic regulators in hematopoiesis has become critically important, because recurrent mutations or aberrant expression of these genes has been identified in both myeloid and lymphoid hematological malignancies. We found that PRMT4, a type I arginine methyltransferase whose function in normal and malignant hematopoiesis is unknown, is overexpressed in acute myelogenous leukemia patient samples. Overexpression of PRMT4 blocks the myeloid differentiation of human stem/progenitor cells (HSPCs), whereas its knockdown is sufficient to induce myeloid differentiation of HSPCs. We demonstrated that PRMT4 represses the expression of miR-223 in HSPCs via the methylation of RUNX1, which triggers the assembly of a multiprotein repressor complex that includes DPF2. As part of the feedback loop, PRMT4 expression is repressed posttranscriptionally by miR-223. Depletion of PRMT4 results in differentiation of myeloid leukemia cells in vitro and their decreased proliferation in vivo. Thus, targeting PRMT4 holds potential as a novel therapy for acute myelogenous leukemia.

The Role of Human Equilibrative Nucleoside Transporter 1 on the Cellular Transport of the DNA Methyltransferase Inhibitors 5-Azacytidine and CP-4200 in Human Leukemia Cells

The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.

Notch activation inhibits AML growth and survival: a potential therapeutic approach

Although aberrant Notch activation contributes to leukemogenesis in T cells, its role in acute myelogenous leukemia (AML) remains unclear. Here, we report that human AML samples have robust expression of Notch receptors; however, Notch receptor activation and expression of downstream Notch targets are remarkably low, suggesting that Notch is present but not constitutively activated in human AML. The functional role of these Notch receptors in AML is not known. Induced activation through any of the Notch receptors (Notch1-4), or through the Notch target Hairy/Enhancer of Split 1 (HES1), consistently leads to AML growth arrest and caspase-dependent apoptosis, which are associated with B cell lymphoma 2 (BCL2) loss and enhanced p53/p21 expression. These effects were dependent on the HES1 repressor domain and were rescued through reexpression of BCL2. Importantly, activated Notch1, Notch2, and HES1 all led to inhibited AML growth in vivo, and Notch inhibition via dnMAML enhanced proliferation in vivo, thus revealing the physiological inhibition of AML growth in vivo in response to Notch signaling. As a novel therapeutic approach, we used a Notch agonist peptide that led to significant apoptosis in AML patient samples. In conclusion, we report consistent Notch-mediated growth arrest and apoptosis in human AML, and propose the development of Notch agonists as a potential therapeutic approach in 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.

FLT3-ITD+ AML Blast, Progenitor and Stem Cell Populations Demonstrate Higher Sensitivity to the Hsp90 Inhibitor PU-H71,

Abstract 3500Hsp90, one of the best-characterized molecular chaperones, plays indispensable roles in folding and assembly, intracellular transport, stabilization, and degradation of proteins, and therefore, facilitating cell signaling. Hsp90 is also involved in tumorigenesis by stabilizing oncogenic client proteins. Thus, Hsp90 inhibition has been considered a promising therapeutic strategy for different types of cancer including leukemia. PU-H71 is a novel HSP90 inhibitor with high specificity for oncogenic Hsp90. We investigated the effect of PU-H71 in acute myelogenous leukemia (AML), in particularly, AML stem cells (AML-SCs) that are known to give rise to AML blasts, are refractory to conventional therapies, and thus likely to account for AML relapses.The effect of PU-H71 was evaluated using a panel of 12 leukemia cell lines. Among the 12 leukemia cell lines tested, MOLM-13 and MV4-11 cells were the most sensitive (LD50s 253 nM and 120 nM respectively). Both MV4-11 and MOLM-13 carry FLT3-ITD mutation (occurring in ∼40% AML cases) and MLL translocations (occurring in ∼20% AML cases). Both MLL and FLT3 have been reported as client proteins of Hsp90. However, other leukemia cell lines with MLL rearrangements such as THP-1 and a MLL-ENL cell line derived from MLL-ENL transformed human CD34+ cord blood cells exhibited resistance to PU-H71 treatment (LD50 > 2 μM). The data suggested that FLT3-ITD+ AML samples may display higher sensitivity to Hsp90 inhibition.To confirm the higher sensitivity of FLT3-ITD+ AML cells to Hsp90 targeted therapy, 15 primary AML patient samples (8 FLT3-ITD mutants and 7 wild type FLT3) were treated with increasing concentrations of PU-H71. Cell viability on different cell populations was evaluated using multiparameter flow cytometry at 48 hours after treatment with PU-H71. The average LD50 of PU-H71 in FLT3-ITD+ AML cells was 492 nM (95% CI, 127.636 – 856.364). In contrast, the average LD50 in AML samples with wild type FLT3 was 2.795 μM (95% CI, 1.058 – 4.532). The near 6-fold difference between LD50s for PU-H71 was significant (p=0.0068). Importantly PU-H71 also killed FLT3-ITD+ AML stem and progenitor cells more effectively. Furthermore, PU-H71 treatment decreased the ability to form colonies in FLT3-ITD+ AML specimens more effectively than FLT3 WT AMLs (97.6% and 79.3% decrease relative to control respectively; N=3; P=0.0236). Importantly, PU-H71 had minor toxicity to normal blood mononuclear cells and normal cord blood hematopoietic stem cells.FLT3-ITD+ cell lines and primary AML cells treated with 0.5 μM PU-H71 showed a substantial decrease of phosphorylated forms of Erk1/2, JNK, AKT, p70RSK, NF-κB(p65) and Stat5, which was observed within 4 hours post PU-H71 treatment, whereas the phosphorylation levels of MAPK p38 remained unaffected. Immunoblotting and phosphoflow assays corroborated the inhibition of AKT and Stat5 signaling by PU-H71 in stem and progenitor populations.In summary, FLT3-ITD+ AML cells display a stronger response to PU-H71, suggesting that the FLT3-ITD mutation results in a higher dependency on Hsp90 to stabilize the aberrant signaling elicited by constitutive activation of FLT3. Our data suggests that PU-H71 represents a novel therapy for FLT3-ITD+ AML patients with the potential to ablate AML-SCs. Disclosures:Roboz:EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.

HSP70 Inhibitor, YK5, Ablates Blast, Progenitor and Stem Cell Populations in Primary Acute Myelogenous Leukemia Cells

Abstract 2493Prognosis for acute myelogenous leukemia (AML) with traditional chemotherapeutic agents remains poor, most patients relapse and die of their disease. Studies have shown that AML can be originated and maintained by leukemia stem cells (LSCs) and that a high percentage of phenotypically-defined LSCs correlate to especially poor prognosis. Thus, novel therapies that can eliminate LSCs are needed. Upregulation of HSP70 has been described in human cancer cells and is often associated with chemoresistance and poor prognosis. Therefore, HSP70 represents a novel target for cancer therapy. Due to this, we tested a novel inhibitor of tumor-HSP70, YK5, and found that it can induce potent cell death in AML cells including progenitor and stem cell populations with minimal effects in normal hematopoietic cells.Fifteen primary AML patient samples were treated with YK5 in varying concentrations for 48 hours to determine the LD50 of YK5. The viability of these samples was determined by multiparameter flow cytometry using cell surface markers to distinguish, lymphocytes, blasts, progenitor and stem cell populations in conjunction with Annexin V and 7AAD. Mean LD50 for all samples tested was 4.5μM (CI 95% 2.239–6.853). Three of these samples had very low LD50s below 2.5μM and only one sample demonstrated relative resistance with a LD50 that was greater than 15μM. Notably, progenitor and stem cell populations were also targeted while normal remaining lymphocytes were spared. Furthermore, we found that LSCs in a subset of patient samples treated with YK5 exhibited higher sensitivity to the compound when compared to blast cells. Importantly, treatment of primary AML samples with 5μM YK5 resulted in a 63% decrease in colony forming ability when compared to untreated control (N=5), suggesting that YK5 can impair malignant progenitors. In contrast, normal CD34+ cord blood cells after treatment with 5μM YK5 for 48hrs exhibited a viability of 57% (N=3). Xenotransplant assays are currently ongoing to determine the ability of YK5 to inhibit LSC function. Treatment with YK5 resulted in a decrease in phosphorylation of STAT5, determined by flow cytometry. Taken together, HSP70 inhibition is a promising potential treatment for AML and has the potential to eradicate leukemic stem and progenitor cells while sparing normal hematopoietic cells. Thus, targeting HSP70 in AML appears to hold great promise.*JFR and KKS contributed equally to this project Disclosures:Roboz:EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.

Abstract C170: The combination of statins and dipyridamole is effective preclinically in AML, MM, and breast cancer.

Abstract Statins are drugs that have been utilized for years to treat hyperlipidemia through inhibition of the rate-limiting enzyme of the mevalonate (MVA) pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Preclinical evidence has demonstrated statins to possess anti-cancer properties against a wide range of tumors but not normal cells. Through the use of a chemical library screen, we hypothesize that the identification of compounds which potentiate the anti-cancer effects of statins will uncover novel molecular pathways and/or targets that can be exploited in combination with the MVA pathway to maximize tumor cell death. A pilot 100-compound library, composed of off-patent pharmacologically active drugs clinically used for a wide spectrum of diseases was screened in the multiple myeloma (MM) KMS11 cell line. Dipyridamole (DP), a commonly prescribed anti-platelet agent potentiated the anti-cancer effects of atorvastatin. The DP-statin combination was synergistic and capable of inducing apoptosis in a variety of acute myelogenous leukemia (AML), MM and breast cancer cell lines. The DP-statin combination also induced apoptosis in primary AML patient samples, but was not toxic to normal PBSCs. In an in vivo AML tumor model, the DP-statin combination was found to be effective at inhibiting tumor growth. DP is known to elicit numerous effects, amongst them, phosphodiesterase (PDE) inhibition. In AML cell lines, activators of the PKA pathway including other PDE inhibitors, also induced apoptosis in combination with statins similar to DP. Interestingly, the DP-statin combination prevented the increase of HMGCR, which occurs following statin treatment as part of a classic feedback response. Further mechanistic investigations to determine how DP potentiates statin-induced apoptosis are underway. As both statins and DP are pre-approved for use in humans, off-patent, and readily available, they have the potential to directly impact patient care. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C170.

Autologous T Cells From AML Patients Can Be Effectively Recruited for In-Vitro Lysis of Blasts by a Novel CD33/CD3-Bispecific BiTE Antibody

AbstractAbstract 14Bispecific T cell-engaging (BiTE®) antibodies combine in one polypeptide chain two single chain antibodies, one specific for CD3 on T cells and one for a tumor-associated antigen. The CD19/CD3-bispecific BiTE antibody blinatumomab has shown in phase 1 and 2 clinical trials very high response rates in patients with non-Hodgkin's lymphoma and acute lymphoblastic leukemia. Here, we report on the potential of a novel BiTE antibody targeting CD33, an antigen broadly expressed by myeloid cells including acute myelogenous leukemia (AML) blasts, in redirecting autologous T cells for in vitro lysis of blasts from AML patients.In a first step, the cytolytic potential of the CD33-specific BiTE (CD33 BiTE) was investigated in co-cultures of enriched resting CD8+ T cells from healthy donors and CD33+ leukemic cell lines KG-1 and U-937 as target cells. CD33 BiTE concentrations as low as 0.1 ng/ml (1.8 pM) mediated effective lysis of leukemic cell lines at effector to target (E:T) ratios of 1:1, whereas no lysis was observed with a solely CD3-binding control BiTE antibody. Peripheral CD8+ T cells that were pre-activated in cell culture or CD8+ T cell clones were even more potent in target cell lysis than previously resting T cells. Data obtained with a 51Cr release assay were comparable to those from a flow cytometry-based assay. Next, primary samples from AML patients were co-cultured with mononuclear cells (MNC) from healthy donors at an E:T ratio of 1:1. After 48 hrs of incubation in the presence of 1 ng/ml CD33 BiTE, a decrease in CD33+ AML blasts as well as of CD33+ monocytes was observed when compared to samples with control BiTE or vehicle. The CD33 BiTE induced upregulation of activation markers CD25 and CD69 on the majority of T cells. We furthermore investigated whether T cells from AML patients were capable of mediating lysis of CD33+ leukemia cells by CD33 BiTE. Resting or in vitro pre-stimulated CD8+ T cells were prepared from peripheral blood of newly diagnosed AML patients and tested for lysis of U937 target cells. Redirected T cells from AML patients were capable of eliminating leukemic cells in the presence of CD33 BiTE as effectively as T cells from healthy controls.Finally, we developed a FACS-based assay that allowed studying autologous blast lysis and T cell behaviour using cryo-preserved patient samples. Upregulation of T cell activation markers in cultures of MNC samples from AML patients was evident following addition of 1 ng/ml CD33 BiTE. Fifty five and 85% of CD4+ cells, and 57 and 65% of CD8+ cells expressed CD25 after 24 h and 48 h, respectively, but not with the control BiTE antibody (all <6%). Despite robust T cell activation, only a limited lysis of myeloid blasts was observed, presumably, due to the short incubation periods and low E:T ratios in the range of 1:5-1:21. We therefore investigated whether blast lysis is more effective after prolonged incubation. In the presence of CD33 BiTEs for 6 days, T cell numbers in AML patient samples dramatically expanded; CD8+ cell counts were up 8-fold, and CD4+ cell counts up 11-fold. This was not observed under control conditions. Up to 85% of AML blasts were now lysed. Currently, a larger collection of primary AML patient samples is being analyzed in order to determine an ex-vivo response rate for CD33 BiTE treatment and the impact of the patient samples' E:T ratio and CD33 expression level on blasts on redirected lysis.Taken together, the novel CD33 BiTE effectively engages and activates autologous T cells for the elimination of AML blasts in vitro and may thereby constitute a novel therapeutic option for the treatment of patients with CD33-expressing myeloid leukemia. Disclosures:Aigner: Micromet Inc.: Research Funding. Kischel:Micromet Inc.: Employment, Equity Ownership. Kufer:Micromet Inc.: Employment, Equity Ownership. Baeuerle:Micromet Inc.: Employment, Equity Ownership. Mackensen:Micromet. Inc.: Research Funding. Krause:Micromet Inc.: Research Funding.

Abstract A28: Aberrant c-Myc protein stabilization and Axin1 deregulation in acute leukemia

Abstract The c-Myc oncoprotein is an important regulator of cellular growth, proliferation, differentiation, and apoptosis, and c-Myc expression is commonly increased in cancer. Historically, increased c-Myc expression in hematopoietic cancers has been explained by gene amplification or chromosomal translocations that deregulate its transcription. Recent work from our lab suggests that deregulated phosphorylation of c-Myc associated with disrupted c-Myc degradation constitutes an additional mechanism by which c-Myc can drive cancer. We have previously shown that c-Myc degradation is regulated by hierarchical phosphorylation at 2 conserved sites, serine 62 (S62) and threonine 58 (T58), and is coordinated by the scaffold protein and tumor suppressor Axin1. Building on our previous work showing that in a variety of human leukemia cell lines c-Myc is aberrantly stabilized with altered phosphorylation at T58 and S62, we are examining the prevalence and potential mechanism of increased c-Myc stability in primary acute myelogenous leukemia (AML), T cell acute lymphoblastic leukemia (ALL), and B cell ALL patient samples. Based on our recent work showing that c-Myc protein stability is negatively regulated by Axin1, we are examining mechanisms of Axin1 deregulation in AML and ALL patient samples. Finally, by manipulating Axin1 in both leukemic and non-transformed hematopoietic cell lines, we will investigate Axin1's role in directly controlling c-Myc stability and phosphorylation in hematopoietic lineages and whether Axin1 may be a potential therapeutic target in acute leukemias. Citation Information: Cancer Res 2009;69(23 Suppl):A28.

Homing and invasiveness of MLL/ENL leukemic cells is regulated by MEF2C

AbstractAcute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis, we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8), which induces a myeloproliferation in vivo. Among the targeted genes, we identified Mef2c, encoding a MCM1-agamous-deficiens-serum response factor transcription factor, and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly, several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions, prompting an investigation of the causal interplay. Using a conditional mouse strain, we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however, its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors, providing a mechanistic link to increased homing and motility. Thus, MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype.

Activated Ca2+/Calmodulin-Dependent Protein Kinase IIγ Is a Critical Regulator of Myeloid Leukemia Cell Proliferation

Ca(2+) signaling is an important component of signal transduction pathways regulating B and T lymphocyte proliferation, but the functional role of Ca(2+) signaling in regulating myeloid leukemia cell proliferation has been largely unexplored. We observe that the activated (autophosphorylated) Ca(2+)/calmodulin-dependent protein kinase IIgamma (CaMKIIgamma) is invariably present in myeloid leukemia cell lines as well as in the majority of primary acute myelogenous leukemia patient samples. In contrast, myeloid leukemia cells induced to terminally differentiate or undergo growth arrest display a marked reduction in this CaMKIIgamma autophosphorylation. In cells harboring the bcr-abl oncogene, the activation (autophosphorylation) of CaMKIIgamma is regulated by this oncogene. Moreover, inhibition of CaMKIIgamma activity with pharmacologic agents, dominant-negative constructs, or short hairpin RNAs inhibits the proliferation of myeloid leukemia cells, and this is associated with the inactivation/down-regulation of multiple critical signal transduction networks involving the mitogen-activated protein kinase, Janus-activated kinase/signal transducers and activators of transcription (Jak/Stat), and glycogen synthase kinase (GSK3beta)/beta-catenin pathways. In myeloid leukemia cells, CaMKIIgamma directly phosphorylates Stat3 and enhances its transcriptional activity. Thus, CaMKIIgamma is a critical regulator of multiple signaling networks regulating the proliferation of myeloid leukemia cells. Inhibiting CaMKIIgamma may represent a novel approach in the targeted therapy of myeloid leukemia.