ALBERT

Description: In this study, we characterized nuclear factor κB (NF-κB) subunit DNA binding in chronic lymphocytic leukemia (CLL) samples and demonstrated heterogeneity in basal and inducible NF-κB. However, all cases showed higher basal NF-κB than normal B cells. Subunit analysis revealed DNA binding of p50, Rel A, and c-Rel in primary CLL cells, and Rel A DNA binding was associated with in vitro survival (P = .01) with high white cell count (P = .01) and shorter lymphocyte doubling time (P = .01). NF-κB induction after in vitro stimulation with anti-IgM was associated with increased in vitro survival (P 〈 .001) and expression of the signaling molecule ZAP-70 (P = .003). Prompted by these data, we evaluated the novel parthenolide analog, LC-1, in 54 CLL patient samples. LC-1 induced apoptosis in all the samples tested with a mean LD50 of 2.8 μM after 24 hours; normal B and T cells were significantly more resistant to its apoptotic effects (P 〈 .001). Apoptosis was preceded by a marked loss of NF-κB DNA binding and sensitivity to LC-1 correlated with basal Rel A DNA binding (P = .03, r2 = 0.15). Furthermore, Rel A DNA binding was inversely correlated with sensitivity to fludarabine (P = .001, r2 = 0.3), implicating Rel A in fludarabine resistance. Taken together, these data indicate that Rel A represents an excellent therapeutic target for this incurable disease.

Description: The identification of genes and pathways that are critical for the development and maintenance of leukemia is important for designing new anti-leukemia therapeutics. However, selection of aberrantly regulated genes and pathways and the translation of these features into targeted anti-cancer drugs represent a significant challenge. Studies have characterized the various oncogenic lesions associated with leukemia, and have shown that specific combinations of mutations are sufficient to induce malignancy in model systems. Thus, we sought to identify the gene signature arising from the introduction of cooperating oncogenic mutations into non-malignant cells. We hypothesized that compounds capable of maximally antagonizing the aberrant expression of this subset of genes would represent potential anti-leukemia therapeutics. To test this hypothesis, we turned to the use of murine genetics and chemical genomics. To this end, we adapted mouse systems created by retroviral transduction of primary marrow cells with the BCR-ABL translocation alone or in combination with the NUP98-HOXA9 translocation. This approach creates authentic models of chronic and blast crisis myeloid leukemia (CML and bcCML, respectively), which has been shown to closely resemble human disease. Next, we employed flow cytometry to separate the transduced marrow into purified primitive lin- populations of either normal, BCR-ABL+, or BCR-ABL+/NUP98-HOXA9+ cells. Importantly, lin- cells were used to focus specifically on those genes most relevant to leukemic stem and progenitor populations. Microarray gene expression profiling was performed on these purified populations in six independent experiments using the Affymetrix Mouse 430 2.0 platform. Gene expression signatures for each transduced population relative to normal lin- cells were obtained. Next, in silico screening for agents that antagonize these signatures was performed comparing the resulting signatures to a local microarray compendium that included the CMap dataset. Gene expression signatures arising from the introduction of BCR-ABL alone identified traditional chemotherapeutics including cytarabine, etoposide, idarubicin – agents considered less effective against leukemic stem cell populations – as well as agents acting along the PI3 kinase pathway. Surprisingly, drugs shown to antagonize the gene signature derived by the introduction the two oncogenic mutations (BCR-ABL and NUP98-HOXA9) included 15-delta-prostaglandin-J2, celastrol, MG- 132, and parthenolide. Each of these drugs eradicate malignant stem and progenitor cells, while sparing normal cells. Thus, the murine-derived signature successfully identified drugs highly relevant to human disease. Our studies therefore demonstrate the utility of combining gene expression-based drug screening and mouse cancer genetics to reveal novel anti-leukemia compounds in humans. DCH and JMA contributed equally.

Description: Abstract 388 Leukemia stem cells (LSCs) have been shown to initiate and maintain AML. Given that LSCs have also been shown to be chemoresistant relative to the bulk leukemia population, LSCs are thought to provide a surviving reservoir of cells that drive disease relapse. This concept is further supported by studies suggesting that poor prognosis is associated with leukemias presenting with a higher percentage of LSCs. Thus, identification of new therapeutic regimens that target LSCs appears to be of clinical significance. Our previous efforts to target LSCs have demonstrated the capability of parthenolide (PTL), and its water-soluble clinical derivative, dimethylamino-parthenolide (DMAPT), to impair the survival and leukemogenic activity of phenotypically- and functionally-defined LSCs, respectively. In order to improve the clinical utility of PTL–based compounds, we sought to determine rational drug combinations that would enhance the efficacy of these agents in vivo. To this end, we turned to the use of chemical genomic screening. The transcriptional signature arising when primary patient specimens are exposed to PTL for 6 h reveals a significant “protective response” (i.e. augmentation of detoxifying enzymes, antioxidant responses, and the unfolded protein response). Given this observation, we hypothesized that compounds capable of impairing the protective response would synergize with PTL and its derivatives and thereby enhance their anti-leukemia activity. To test this hypothesis, we interrogated the Connectivity Map database for instances of compounds that produced maximal inhibition of the protective response at the gene expression level. Overwhelmingly, this screen indicated compounds acting along the PI3 kinase pathway including wortmannin, LY-294002, and rapamycin. Indeed, exposure of primary AML cells to the combination of wortmannin and sub-lethal doses of PTL significantly decreased viability of AML cells (24% viable) relative to PTL (65% viable) or wortmannin (66% viable) alone. Moreover, the combination resulted in a significant decrease in colony formation (Wort=67% , PTL=87.5 and PTL+Wort = 6% CFU relative to untreated). The effect of these combinations is synergistic and not additive (Chou-Talay method). Importantly, these effects remained confined to AML cells and not their normal hematopoietic counterparts. Furthermore, these observations were corroborated with rapamycin and temsirolimus. To examine molecular mechanisms underlying the enhanced anti-leukemia activity, immunoblot analyses were performed and demonstrated that the PTL-induced protective response was abolished by the combination of PTL with wortmannin or rapamycin, consistent with the chemical genomic prediction. Finally, to assess in vivo activity of the drug combination we employed the clinical agents DMAPT and temsirolimus in a mouse xenotransplant model. Briefly, NOD/SCID mice were injected with primary human AML cells and at three weeks post transplant began a three-week course of daily treatment (vehicle control, DMAPT alone, temsirolimus alone, or DMAPT+temsirolimus). Animals were sacrificed after treatment (i.e. six weeks post transplant) and tumor burden was evaluated in the bone marrow. The mean percent of leukemic cells for the combination of temsirolimus and DMAPT treatment resulted in a four-fold decrease which was significant when compared to either treatment alone (P

Description: Abstract 3966 Poster Board III-902 Since oncogenic activation of HoxA9 is induced by multiple chromosomal translocations affecting MLL1 (11q23)(e.g. MLL-Af9) or Nup98 (11p15)(e.g. Nup98-HoxA9 or Nup98-Nsd1), the function of the HoxA9 transcription factor is of critical interest in human acute myeloid leukemia (AML). HoxA9 forms heterodimeric DNA binding complexes with members of the Pbx and/or Meis family of homeodomain proteins. Importantly, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. In a transcriptional circuit conserved to Drosophila, we have recently shown that Gfi1 represses HoxA9, Meis1 and Pbx1 expression, that Gfi1 and HoxA9 demonstrate dramatic epistatic relationships, and that Gfi1 loss of function is potently preleukemic. Our new bioinformatic, biochemical and expression data reveal microRNA genes to be targets of endogenous HoxA9 versus Gfi1 antagonism. Moreover, these miR are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts oncogenic signaling by HoxA9, Nup98-HoxA9 and MLL-Af9 (but not AML-ETO which does not signal through HoxA9). In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia lethality. These data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins. Disclosures: No relevant conflicts of interest to declare.

Description: CLL-1 (C-type Lectin-Like Molecule-1) is an inhibitory receptor expressed on myeloid cells which was previously shown to be expressed on AML cancer stem cells. To further validate the potential therapeutic against CLL-1, we generated a series of monoclonal antibodies (mAbs) against CLL-1 and assessed their cytotoxic and anti-cancer activities. While expression of CLL-1 was restricted to myeloid cells, it was expressed in 80% (37/46) of AML blasts but not in ALL blasts (n=5). Expression on AML CD34+/CD38- stem cells was detected in 71% (12/17) of cases. Selected anti-CLL-1 mAbs mediated dose-dependent. Complement Dependent Cytotoxicity (CDC) against various AML-derived cell lines with no detectable cytotoxic activity against lymphoid derived cell lines. Moreover, human embryonic kidney 293 cells became susceptible to anti-CLL-1 mAb mediated killing only after transfection with CLL-1, demonstrating that cytotoxic activity is mediated through a specific interaction with CLL-1. Furthermore, anti-CLL-1 mAbs showed CDC activity against all AML blasts tested in ex vivo assays (n=13), while no activity was observed against ALL blasts. CLL-1 efficiently internalizes upon antibody binding in both 293 transfected cells as well as AML cell lines, demonstrating the potential therapeutic opportunity for toxin-conjugation of anti-CLL-1 mAbs. In vivo anti-cancer activity of the lead chimeric mAb against CLL-1 was tested in an HL-60 xenograft model. In this model, growth of established tumors was reduced by 38% at 5 mg/kg twice weekly dosing. Our results demonstrate CLL-1 as an attractive target for AML with restricted expression on cells from myeloid origin, AML blasts and leukemic stem cells, as well as specific cytotoxic activity in in vitro, ex vivo and in vivo assays. We are currently undertaking additional xenograft models to evaluate the therapeutic potential of these mAbs against primary AML engrafted cells and in combination with chemotherapy in vivo.

Description: Previous studies have demonstrated that the plant-derived compound parthenolide (PTL) induces AML stem cell specific apoptosis while sparing normal counterparts. We have observed that PTL strongly inhibits NF-kappaB activity. Moreover, anti-oxidants such as N-acetylcysteine block the apoptotic process. However, the molecular events involved in the process are not completely understood. Given the demonstrated efficacy of PTL at targeting AML stem cells (AML-SCs), we hypothesized that a molecular signature that captures events invoked during PTL-induced apoptosis will provide a powerful tool for improving AML-SC targeting. To this end, we have conducted pharmacogenomic studies using primary CD34-enriched cells obtained from 12 randomly selected AML specimens. To capture dynamics of the PTL response, primary CD34+ AML were cultured in vitro and exposed to 5 micromolar PTL for either 1 hour or 6 hours. Labeled mRNA was hybridized to Affymetrix HG-U133plus2 chips. Differential expression of individual genes was assessed using moderated and permutation-based paired tests with false discovery rate control. Additionally, to capture pathway-level events possibly representing drugable targets, we employed functional, network, and robust geneset-based approaches to mine the data for coordinated changes in the expression of groups of biologically related genes, e.g., those controlled by a common transcription factor. By 1 hour post-treatment, we were able to detect the beginning stages of a cascade of responses leading to activation of antioxidant enzymes, cytoskeletal reorganization and downregulation of adhesion molecules, modulation of protein synthesis, proteasome remodeling, and modulation of cell cycle progression genes (q 〈 0.05). Transcription factor (TF) binding motif enrichment studies show that several of these processes are governed by the involvement of transcription factors such as Nrf2, NFkappaB, and interferon regulatory factor-1 (IRF-1) and their respective upstream controllers. The biological relevance of the array findings was directly validated in independent studies demonstrating heightened oxidative state and the associated nuclear translocation of Nrf2 and upregulation of its target heme oxygenase I. We further hypothesized that a number of genes activated immediately following PTL exposure are important for promoting AML survival and/or drug resistance and that such genes would represent potentially useful drug targets. This rationale resulted in our selection of the target, eukaryotic initiation factor 5 (eIF5). Our array-based analyses revealed that the eIF5 gene is upregulated within 1 h of PTL exposure and is further upregulated at 6 h. eIF5 activation is regulated by hypusination and can be inhibited by the anti-fungal agent, ciclopirox. Therefore, we tested the biological effects of 2.5 micromolar PTL alone or in combination with ciclopirox on independent primary AML specimens. Exposure to either drug alone had a negligible effect on primary normal bone marrow or AML cells after 16 h growth in suspension culture. However, the combination of both resulted in ~20% viability for AML relative to ~80% viability for normal bone marrow. Importantly, the combination of PTL and ciclopirox completely eradicated the colony forming ability of AML cells while only modestly affecting normal bone marrow. These studies support the utility of high-throughput genomic studies as a conduit for drug discovery.

Description: Leukemia stem cells (LSCs) have been shown to play a crucial role in the pathogenesis of acute myelogenous leukemia (AML). However, current chemotherapy agents have not demonstrated effective targeting of the LSC population. Thus, failure to eradicate LSCs may lead to disease relapse and progression. Consequently, the identification and characterization of drugs that can efficiently eradicate the LSC population is required. We have identified a compound, 4-benzyl, 2-methyl,1,2,4-thiadiazolidine,3,5 dione (TDZD-8), with the novel characteristic of rapidly inducing cell death of different types of leukemia cells, including AML progenitor and stem cell populations. After 18–24 hours of treatment with 20 uM TDZD-8, the mean survival for primary AML cells was 7% (n=30), blast crisis CML 5% (n= 5), CLL 3% (n=13) and ALL 6% (n= 4). In contrast, normal specimens treated in the same fashion showed 80% mean survival (n=10). Functional assays were performed to determine the effect of TDZD-8 on primitive populations. NOD/SCID xenotransplant analysis showed a 93% decrease of engraftment for AML specimens, while normal cell engraftment was only reduced by 11%. Furthermore, colony-forming assays demonstrated a 93% decrease in CFU formation for AML cells (n=11, where 6 of the specimens showed no CFUs), in contrast to a 30% decrease in normal cells. Strikingly, cell death occurred with extremely rapid kinetics. Time course studies from the different primary AML specimens demonstrated that most of the cell death can be observed within 2 hours after exposure to TDZD-8 (where 10% of the samples tested appear dead with as little as 15–30 minutes of drug exposure). Furthermore, a 30 minute exposure to the drug was sufficient to irreversibly induce cell death of primitive CD34+CD38- cells and CFUs. Interestingly, TDZD-8 was originally described as a GSK-3b inhibitor and analogs to this molecule have also been found to activate PPAR-gamma. However, when we tested different known GSK-3b inhibitors (n=12) and PPAR-gamma activators (containing a thioazolidine ring) we did not observe the toxicity, specificity, or kinetics observed with TDZD-8. Molecular studies suggest that the mechanism by which TDZD-8 induces cell death involves a rapid thiol depletion, Nrf2 activation, and NF-kappaB inhibition. Moreover, a rapid loss of membrane integrity was observed using imaging flow cytometry (Amnis ImageStream) to determine permeability of different viability dyes. Furthermore, TDZD-8 induced cytochrome c release, but cell death was not blocked by pan-caspase inhibitors, suggesting a non-apoptotic cell death mechanism. Finally, preliminary in vivo experiments using mice transplanted with primary AML cells showed that Nrf2 was induced as soon as 1h after dosing animals with 4.4 mg/kg of TDZD-8 (i.p.). Together, these data suggest that TDZD-8 represents a novel class of anti-leukemia specific molecules capable of destroying leukemia progenitor and stem cells with extremely rapid kinetics.

Description: We have previously demonstrated that Parthenolide (PTL), the principal component of the medicinal plant Feverfew, selectively induces apoptosis in AML stem cells while sparing normal counterparts. Recent reports show that PTL is also effective against CLL cells. Additionally, we observed PTL efficacy for ALL cells in vitro. These findings suggest that PTL-derived drugs may provide a unique means of leukemia therapy. A clinical study using Feverfew showed PTL plasma levels were not sufficient to achieve the concentration needed for AML targeting as established by our in vitro studies. Therefore, we synthesized a family of PTL derivatives designed to be more water-soluble. An amino analog, LC-1, retained the anti-leukemia properties of PTL with 1000× improved water solubility. In vitro studies indicate that LC-1 induces irreversible apoptosis of primary human AML cells within 8 h of treatment. Analysis of phenotypically primitive cells (CD34+CD38−, n=27) treated with 5.0–7.5 uM LC-1 for 18–24 h demonstrated an average cell kill of 85–90%. In contrast, normal CD34+CD38− cells showed less than 10% death under the same conditions. Similarly, colony-forming potential of AML specimens (n=5) was inhibited by 〉90% but by

Description: Abstract 2734 Poster Board II-710 We have previously demonstrated that parthenolide (PTL), a naturally occurring small molecule found in feverfew Chrysanthemum parthenium, induces apoptosis in primary acute myeloid leukemia (AML) cells, including the stem and progenitor cell compartment. Based on these preclinical findings, a PTL derivative (dimethylamino parthenolide) is currently being evaluated in a phase I clinical trial. However, despite the promising activity of PTL, its underlying mechanism of action remains poorly understood. Thus, we have undertaken biochemical studies to better characterize how PTL mediates leukemia-specific cell death. Chemically, the key structural feature of PTL is its alpha-metheylene-gamma-lactone moiety, which via Michael reaction is predicted to mediate potent free thiol scavenging, an activity readily observed in PTL-treated cells. Reported consequences of PTL chemical reactivity in a variety of cell types, represent a broad range of activities that include inhibition of NFkB, activation of p53, ubiquitination of MDM2, inhibition of DNMT1 and inhibition of HDAC1. To better define the specific activities responsible for parthenolide-mediated leukemia cell death, we have employed two general approaches. First, we generated a biotinylated analog of parthenolide (PTL-biotin), which was shown to retain the anti-leukemia activity of the parent compound. PTL-biotin was then used in biochemical pull-down assays to purify parthenolide target proteins, followed by liquid phase chromatography-mass spectrometry (LC-MS) for protein identification. To further verify molecular interactions, native non-biotinylated parthenolide was used to compete binding between candidate targets and PTL-biotin. These studies identified HSP70 as a direct target of PTL. Notably, cysteine-17 of HSP70 is exposed to the ADP/ATP binding site crevice and a molecular docking study indicates that the covalent attachment of PTL to this residue should disrupt the ATP hydrolysis function of the protein. These findings imply that inhibition of HSP70 may contribute to the cell death mechanism underlying PTL anti-leukemia based activity. As a second approach to characterizing PTL, we have performed comparative studies using the closely related compound costunolide (CSN). Since previous structure-activity studies with PTL analogs revealed that opening of the epoxide ring at C4-C5 of the molecule completely destroys the anti-tumor activity, we sought to utilize a compound lacking this feature. CSN lacks the epoxide group, but is otherwise identical to PTL, and retains the key alpha-metheylene-gamma-lactone moiety. Interestingly, at concentrations where PTL is highly cytotoxic, CSN does not induce leukemia-specific cell death (less than 10% death for primary AML cells at 7.5 microM). Analysis of CSN activity demonstrated that despite the lack of AML cell death, CSN still induced loss of free thiols and increased reactive oxygen species in a fashion comparable to PTL (as measured by mBBR and CM-H2DCFDA based flow cytometry). However, CSN is markedly less effective as an inhibitor of NFkB activity (measured by phosphorylation level of NFkB p65). Taken together these findings indicate that oxidative stress alone is not sufficient for PTL-mediated cell death, and further extend previous molecular genetic data demonstrating that NFkB inhibition is an important component of the overall cell death mechanism. The data also show that the alpha-metheylene-gamma-lactone moiety alone is not sufficient to mediate all aspects of PTL activity, and that at least some activity/specificity is created by juxtaposition of the epoxide group. Based on these studies, as well as previous data, we propose that inhibition of NFkB and HSP70 are components of the parthenolide-mediated cell death mechanism, and that oxidative stress is a necessary but not sufficient aspect of its leukemia-specific activity. Disclosures: No relevant conflicts of interest to declare.

Description: Leukemia is thought to arise from malignant stem cells, which have been described for acute and chronic myeloid leukemia (AML and CML) and for acute lymphoblastic leukemia (ALL). Leukemia stem cells (LSCs) are relatively resistant to current chemotherapy and likely contribute to disease relapse and progression. Consequently, the identification of drugs that can efficiently eradicate LSCs is an important priority. In the present study, we investigated the antileukemia activity of the compound TDZD-8. Analysis of primary AML, blast crisis CML (bcCML), ALL, and chronic lymphoblastic leukemia (CLL) specimens showed rapid induction of cell death upon treatment with TDZD-8. In addition, for myeloid leukemias, cytotoxicity was observed for phenotypically primitive cells, in vitro colony-forming progenitors, and LSCs as defined by xenotransplantation assays. In contrast, no significant toxicity was observed for normal hematopoietic stem and progenitor cells. Notably, cell death was frequently evident within 2 hours or less of TDZD-8 exposure. Cellular and molecular studies indicate that the mechanism by which TDZD-8 induces cell death involves rapid loss of membrane integrity, depletion of free thiols, and inhibition of both the PKC and FLT3 signaling pathways. We conclude that TDZD-8 uses a unique and previously unknown mechanism to rapidly target leukemia cells, including malignant stem and progenitor populations.