The Small Molecule Inhibitor of VLA4 TBC3486 Sensitizes Resistant ALL to Chemotherapy

Abstract Abstract 1500 Significant progress notwithstanding, drug resistant acute lymphoblastic leukemia (ALL) remains a therapeutic challenge, as well as acute and long-term off-target toxicity of anti-ALL therapies can be dose-limiting or debilitating. Therefore, the development of more targeted therapies is desirable. We recently provided evidence that chemotherapy resistance of ALL cells can be partly overcome by interfering with the function of VLA4, the alpha4beta1 integrin, in vivo. In those studies, we used the anti-functional antibody Natalizumab. We extended our studies to an alternative VLA4 inhibitor, the novel non-peptidic small molecule TBC3486. Previous in vitro assays and molecular modeling studies indicate that TBC3486 behaves as a ligand mimetic, competing with VCAM-1 for the MIDAS site of VLA-4. As such, the compound has been shown to be efficacious in VLA-4 dependent models of inflammatory and autoimmune disease. The potential usefulness of this novel inhibitor in leukemia treatment was tested in our established in vitro and in vivo assays. LAX7R cells, primary pre-B-ALL with a normal karyotype from a patient with an early relapse, were used throughout for the studies reported here. LAX7R cells were treated with 25μM TBC3486 or THI0012 control, the inactive enantiomer of TBC3486, and seeded onto plates coated with human VCAM-1. Adhesion, scored after 2 days, was significantly inhibited by TBC3486 compared to control treated cells (7.9%±4.0 vs 95.4%±8.0; p=0.003). Proliferation rate and cell viability were unaffected by the treatments. In a co-culture system of LAX7R cells with OP9 stroma cells, which we use as an in vitro model of stroma-mediated chemotherapy resistance, we assessed differential effects of VDL (Vincristine, Dexamethasone, L-Asparaginase) on leukemia cell survival in the presence or absence of TBC3486. Stromal adhesion significantly protected LAX7R cells against VDL chemotherapy; this effect was significantly attenuated by TBC3486 compared to the control as determined by Trypan blue exclusion of dead cells (Cell viability of 39.9%±5.1 vs. 57.2±1.8; p=0.02). After these encouraging observations, we next evaluated the benefit of TBC3486 on leukemia progression in a xenotransplant assay. LAX7R cells were lentivirally labelled with luciferase for in vivo tracking and injected into NOD/SCID hosts. Three days after leukemia cell transfer, mice received either TBC3486 or THI0012 (control) (10mg/kg/d) daily for 2 weeks (intraperitoneally), with or without VDL chemotherapy. This experiment is in progress, but already survival of leukemia-bearing mice was significantly prolonged, from a median survival time (MST) for control mice of 33 days post-leukemia injection to a MST of 47 days post-leukemia injection for TBC3486 treated mice (p=0.02). Similarly, bioluminescence imaging revealed a marked delay of leukemia cell dissemination (p<0.0001). Taken together, our data demonstrate that small molecule inhibition of VLA4 using the novel TBC3486 is a suitable approach for targeting of chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant small molecule inhibition of integrins to overcome relapse of ALL. Disclosures: No relevant conflicts of interest to declare.

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Interrogating Novel Bromodomain Inhibition Resistance Mechanism in Mllr Leukemia

Blood ◽

10.1182/blood-2021-147003 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 26-26

Author(s):

Chunliang Li ◽

Shaela Wright ◽

Jianzhong Hu ◽

Yang Zhang ◽

Judith Hyle ◽

...

Keyword(s):

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Adaptor Protein ◽

Leukemia Cells ◽

Resistance Phenotype ◽

Bet Inhibitors ◽

Resistant Cells

Abstract MLL-rearranged (MLLr) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLLr patients achieved an initial remission but ultimately relapsed. Bromo- and Extra-Terminal domain inhibitors (BETi) prevent the progression of many cancer types in preclinical studies, including MLLr leukemia. However, the mechanisms controlling drug response and resistance of BET inhibitors are not well understood. We have addressed this timely, crucial scientific question by completing genetic screens to explore potential BETi resistance mechanisms. By conducting genome-wide and targeted loss-of-function CRISPR screens using MLLr AML cell lines upon BETi treatment including ABBV-744, JQ1, and dBET1, we discovered that Speckle Type POZ (SPOP) gene deficiency leads to significant BETi resistance in in vitro cell culture systems (SEM, OCI-AMl2 and MV4,11), and by in vivo transplantation of human MLLr leukemia SEM cells into immune-deficient mice. However, no BETi resistance phenotype was seen in non-MLLr SPOP-deficient cells. SPOP was previously reported as an adaptor protein to bridge the E3 ubiquitination complex component CUL3 to the substrate proteins BRD4 and MYC in prostate and many other solid cancers. However, in SPOP knockout MLLr leukemia cells, TRIM24, not BRD4 and MYC, was identified as a substrate likely responsible for SPOP's role in drug resistance. Genetically blocking TRIM24 via CRISPR knockout in SPOP-knockout cells reversed the BETi resistance phenotype. Transcriptomic analysis of TRIM24-deficient cells identified the GSK3A signature as the top influenced pathway. Additionally, proteomics expression analysis and a kinase vulnerability CRISPR screen also indicated that resistant cells are sensitive to GSK3B inhibition. Further validation by CRISPR knockout and pharmaceutical blockage of GSK3A/3B (by ChIR-98014) sensitized the SPOP-deficient resistant cells to BETi treatment in vitro. In SEM xenograft models in NSG mice, ABBV-744 or CHIR-98014 minimally impacted human CD45 + leukemia cell proliferation while synergistic treatment significantly reduced the tumor progression. In summary, our data suggest the novel SPOP/TRIM24/GSK3A/3B axis plays an essential role in BETi therapy-resistant leukemia cells. Targeting GSK3A/3B pathways by ChIR-98014 can overcome SPOP-associated BETi resistance in in vivo preclinical models of MLLr leukemia. Successful outcomes following combination therapy using ChIR-98014 and BETi in PDX models would translate to a clinical application that holds the promise to cure MLLr leukemia. Disclosures No relevant conflicts of interest to declare.

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Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.810.810 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 810-810 ◽

Cited By ~ 3

Author(s):

Haiying Qin ◽

Sang M Nguyen ◽

Sneha Ramakrishna ◽

Samiksha Tarun ◽

Lila Yang ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Chain ◽

Dual Targeting ◽

Single Antigen ◽

Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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Unique Thrombopoietic Activity of Novel PKC Agonist Ingenol 3,20 Dibenzoate.

Blood ◽

10.1182/blood.v114.22.3622.3622 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3622-3622

Author(s):

Frederick Karl Racke ◽

Maureen E Baird ◽

Rolf Barth ◽

Tianyao Huo ◽

Weilian Yang ◽

...

Keyword(s):

Conflicts Of Interest ◽

Leukemic Cell ◽

The Novel ◽

Drug Induced ◽

Platelet Recovery ◽

Platelet Production ◽

Platelet Counts ◽

Pkc Isoforms

Abstract Abstract 3622 Poster Board III-558 Despite recent advances in our understanding of megakaryocytic growth and platelet production, thrombocytopenia remains a difficult problem in the clinical management of patients with hematologic malignancies. Thrombopoietin (TPO) is the major cytokine involved in the normal production of platelets. However, the use of TPO has been relatively unsuccessful for the treatment of these patients and platelet transfusions remain the primary treatment for thrombocytopenia despite their significant cost and relatively short-lived responses. Thus, there remains an important clinical need for the development of novel approaches to generate platelets. Despite numerous reports on protein kinase C (PKC) agonists as promoters of megakaryocytic differentiation in leukemic cell lines and primary cells, little is known about their in vitro effects on primary CD34-selected progenitors or when administered in vivo. In the present study, we examine that effects of the novel PKC isoform agonist ingenol 3,20 dibenzoate (IDB) on megakaryocyte differentiation from CD34+ cells cultured in TPO and stem cell factor (SCF) or erythropoietin/SCF and its effects on platelet production in BALB/c mice. IDB potently stimulates early megakaryopoiesis and redirects the specificity of EPO to favor megakaryopoiesis over erythropoiesis. In contrast, broad spectrum PKC agonists such as phorbol myristate acetate, mezerein, and indolactam V fail to promote megakaryopoiesis. In vitro, IDB stimulates early expression of the promegakaryopoietic transcription factors egr1 and fli-1 and downregulates the proerythropoietic factors KLF1 and c-myb. Induction of the early megakaryocytic marker, CD9, was observed within the first 24 hrs of treatment with IDB and CD9 induction was blocked by the PKC inhibitor bisindolylmaleimide, which inhibits both novel and conventional PKC isoforms. In contrast, an inhibitor of conventional PKC isoforms, Gö6976, failed to block CD9 induction. In vivo, single intraperitoneal injections of IDB selectively increased platelet counts in BALB/c mice by 50% (plt= 630,000 vs. 985,000/μl; p<.005) at day 7 without affecting hemoglobin (Hgb) concentration or white counts (WBC). Mice treated with low dose radiation (2-4 Gy) had a transient drop in both platelet and WBC counts. Pretreatment with IDB 3 hrs prior to irradiation increased the platelet counts without improving WBC. More severe radiation exposure (6-8 Gy) causes pancytopenia. IDB treatment 3 hrs prior to 6 Gy irradiation significantly reduced the thrombocytopenia (plt=192,000 vs 594,000/μl; p<0.005) and anemia (hemoglobin=11.9 vs. 13.5gm/dl); p<0.005) without affecting the drop in WBC (WBC=1,200 vs. 1,300/μl; p=NS) at 14 days following irradiation. For mice treated with 8 Gy radiation, IDB pretreatment resulted in similar improvements in platelet counts (plt=111,000 vs. 443,000/μl; p<0.005) and hemoglobin (hgb=8.2 vs. 12.7 gm/dl; p<0.005) at 21 days following irradiation. The mitigation of thrombocytopenia is accompanied by marked increases in the megakaryocyte content in both the spleens and bone marrows of IDB-treated mice. Most importantly, IDB mitigated radiation-induced thrombocytopenia, even when administered 24 hrs after irradiation (plt=80,000 vs. 241,000/μl at 14 days following 6 Gy irradiation; p<0.01). Finally, IDB improved the survival of lethally irradiated mice. Our data suggest that the novel PKC isoform agonist IDB promotes the early differentiation of megakaryocytes from hematopoietic progenitors at the resulting in a significant improvement in platelet recovery following irradiation. IDB also improved Hgb levels following higher radiation doses. This may be due to improved hemostasis secondary to increased platelet numbers; however, an additional radioprotective effect on erythroid precursors cannot be excluded. These results strongly support our hypothesis that the novel PKC agonist IDB may be useful for the treatment of radiation and possibly drug-induced thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

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Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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Immunornase Therapy Of Lymphoma With Novel CD22-Targeting Quadruple Ranpirnase

Blood ◽

10.1182/blood.v122.21.3075.3075 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3075-3075

Author(s):

Donglin Liu ◽

Thomas M Cardillo ◽

David M Goldenberg ◽

Chien-Hsing Chang

Keyword(s):

Cell Lines ◽

Targeted Delivery ◽

Stock Options ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Xenograft Model ◽

Stock Option ◽

Amino Acid Sequences

Abstract Ranpirnase (Rap) is an amphibian ribonuclease showing anti-tumor activity in clinical studies. We have previously reported that targeted delivery of Rap by chemical conjugation or recombinant fusion with antibodies specific for CD22, CD74 and Trop-2 could enhance its in vitro cytotoxicity as high as 10,000-fold in selected malignant cell lines. The DOCK-AND-LOCKTM (DNLTM) platform technology is a powerful method to construct novel agents of defined composition and retained bioactivity by site-specific conjugation of two types of modules, one containing the dimerization and docking domain (DDD) of cAMP-dependent protein kinase A (PKA), referred to as the DDD module, and the other bearing the anchoring domain (AD) of an interactive A-kinase anchoring protein (AKAP), referred to as the AD module. Among the distinctive features of DNL are the spontaneous formation of a dimer of the DDD module and the self-assembly of the DDD module with the AD module into a non-covalent complex, which is subsequently rendered covalent with disulfide bonds to enhance stability in vivo. The amino acid sequences of a pair of DDD and AD linkers useful for the DNL conjugation are termed DDD2 and AD2, respectively. To further explore the potential of Rap-based immunotoxins, we expressed a DDD2-module of Rap in E. coli and linked the resulting dimer of Rap to an AD2-module of a humanized IgG (expressed in myeloma cells) at each of the carboxyl termini of either the light chain (the CK-format) or the heavy chain (the CH3-format), thus producing a class of novel immunoRNases with quadruple Rap. To date, we have evaluated a pair of such constructs, 22* -Rap and 22-Rap, comprising four copies of Rap linked to the CK or CH3 termini of epratuzumab (humanized anti-CD22), respectively, in a panel of CD22-expressing human lymphoma/leukemia cell lines, which include Burkitt lymphoma (Daudi, Raji, Ramos), acute lymphoblastic leukemia (REH, 697, and RS4;11), and mantle cell lymphoma (Granta-519 and Jeko-1). The results of the MTS assay indicated that 22* -Rap was highly active (EC50 ≤ 1 nM) against Daudi, Ramos, Raji, REH, 697, RS4;11, and Granta-519 cells. Comparable cytotoxicity with EC50 values in the subnanomolar range also was observed for 22-Rap in Ramos, Daudi and Jeko-1 cells. In contrast, neither the individual DNL components (IgG-AD2 or Rap-DDD2), tested alone or in combination, nor E1* -Rap, the counterpart of 22* -Rap, generated by substituting epratuzumab with hRS7 IgG (humanized anti-Trop-2), showed notable cytotoxicity. In a disseminated Daudi xenograft model in which treatment with 10 or 20 µg of 22* -Rap (q4dx4) started 7 days after intravenous inoculation of the tumor cells, all 10 mice (5 in each group) survived over 126 days and were tumor-free, whereas the control groups, treated with saline, epratuzumab (25 µg, q4dx4), or the same dose-schedule of nonspecific control, E1* -Rap, succumbed within 36 days. These promising results, together with the findings that 22* -Rap at 10 nM was marginally toxic to B cells and other hematological cells in PBMCs, encourage further development of 22* -Rap or 22-Rap for therapy of CD22-expressing lymphomas and leukemias. Disclosures: Liu: IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other; Immunomedics, Inc.: Employment, Stock option Other. Cardillo:Immunomedics, Inc.: Employment, Stock option Other. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other; Immunomedics, Inc: Employment, Stock option Other.

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UNC569, a Novel Small-Molecule Mer Inhibitor with Efficacy against Acute Lymphoblastic Leukemia In Vitro and In Vivo

Molecular Cancer Therapeutics ◽

10.1158/1535-7163.mct-13-0040 ◽

2013 ◽

Vol 12 (11) ◽

pp. 2367-2377 ◽

Cited By ~ 35

Author(s):

Sandra Christoph ◽

Deborah DeRyckere ◽

Jennifer Schlegel ◽

J. Kimble Frazer ◽

Lance A. Batchelor ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Small Molecule ◽

Lymphoblastic Leukemia

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Targeting Nucleolin for Reversal of Chemotherapy Resistance in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-127073 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5058-5058

Author(s):

Jianda Hu ◽

Yanxin Chen ◽

Zhengjun Wu ◽

Lingyan Wang ◽

Jingjing Wen ◽

...

Keyword(s):

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Drug Sensitivity ◽

De Novo ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Mrna Level ◽

Chemotherapy Resistance

Chemotherapy resistance is considered to be the principal cause of ineffective treatment in acute lymphoblastic leukemia (ALL). Nucleolin (NCL) is high expression andplays oncogenic roles in most cancers. However, less research on the role of NCL in hematologic malignancies was noted. Our previous studies have showed that overexpression of NCL was associated with worse prognosis in the patients with acute leukemia and NCL expressionwashigher in resistant HL-60/ADR than in sensitive HL-60 cells. The potential mechanisms of NCL in chemotherapy resistance have yet to be revealed. Here we presented that expression of NCL was associated positively with chemotherapy resistance and poor prognosis in ALL. Overexpressed NCL at both mRNA and protein level was relevant to a poorer overall survival (OS) and relapse free survival (RFS), indicating NCL as an independent prognostic marker in ALL. mRNA level of NCL in de novo ALL was quantitatively higher than in complete remission(CR) status, and refractory/relapse ALL had the highest level. Upon above clinical data, we further investigated the mechanism(s) by which NCL regulated drug resistance in ALL cells. Remarkably, NCL expression was higher in resistant ALL cells relative to sensitive parental cells. When treated with ADM, NCL level was decreased in sensitive parental cells while unchanged in resistant cells. Overexpressing NCL suppressed drug sensitivity, altered drug effluxion and decreased intracellular drug accumulation, while inhibition of NCL led to a completely reversed appearance, more intracellular Adriamycin(ADM) mean fluorescence intensity (MFI) and percentage of ADM accumulated cells population. Overexpression of NCL increased significantly the IC50 of ADM. The IC50 of ADM on Jurkat-NCL-overexpression(OE), Jurkat-NCL-knockdown(KD), Molt-4-NCL-OE, Molt-4-NCL-KD, Nalm-6-NCL-OE, Nalm-6-NCL-KD were 1.362±0.271μg/ml, 0.077±0.010μg/ml, 4.863±0.733μg/ml, 0.081±0.018μg/ml, 0.237±0.042μg/ml and 0.046±0.002μg/ml, respectively (P <0.05). Involvement of ATP-binding cassette (ABC) transporters was proved in NCL mediated drug resistance. Silencing NCL resulted in a decrease of P-gp, MRP1, LRP and BCRP in ALL cells, and NCL overexpression increased the MRP1, LRP and BCRP. The Akt/mTOR and ERK signaling pathways were involved in this procedure. Notably, co-IP assays confirmed the NCL-Ras, NCL-ERK and NCL-BCRP interaction. For intervention study, aptamer AS1411, a NCL inhibitor, could reduce drug resistance in ALL cell lines and primary ALL cells.Moreover, AS1411 treatment decreased BCRP protein expression. Furthermore, the ALL leukemia models that nude mice engrafted with Nalm-6 cells and NCG mice engrafted with Luc+ Nalm-6 cells were established, then treated with ADM plus AS1411 or control CRO for comparison drug sensitivity and survival. Growth of subcutaneous xenograft tumors was inhibited in those treated with AS1411 or ADM, compared to their respective controls treated with CRO or PBS. The stronger inhibition effect was observed in those treated with AS1411 combined with ADM. For Luc+Nalm-6 derived ALL model, leukemia progression was suppressed in mice treated with AS1411 and AS1411 combined with ADM. AS1411and ADM, especially combination of AS1411 and ADM, could improve survival of the leukemic mice compared to those treated with PBS. The results showed that NCL targeted by AS1411 sensitized ADM treatment and prolonged survival in vivo. In summary, our findings revealed NCL as a survival predictor and the novel role of NCL in ALL chemo-resistance. NCL may be a potential target for improving outcome in ALL. Disclosures No relevant conflicts of interest to declare.

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Adipocytes Secrete Factors That Cause Leukemia Drug Resistance.

Blood ◽

10.1182/blood.v112.11.1346.1346 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1346-1346

Author(s):

James W Behan ◽

Jason P Yun ◽

Marina P Proektor ◽

Ehsan A Ehsanipour ◽

Anna Butturini ◽

...

Keyword(s):

Bone Marrow ◽

Chemotherapy Resistance ◽

Leukemia Cell ◽

Conditioned Media ◽

Leukemia Cells ◽

Cell Contact ◽

Human Leukemia ◽

Bone Marrow Niche

Abstract We have previously shown that obesity is an independent predictor of leukemia relapse in children. We have also shown that obese mice transplanted with syngeneic leukemia cells have poorer survival after chemotherapy, even when they are dosed proportional to body weight. Since interactions between leukemia cells and cells of the bone marrow niche are considered important for chemotherapy resistance and relapse, and adipocytes can comprise ~50% of the bone marrow niche, we developed in vivo and in vitro models to investigate the role of adipocytes in the leukemia microenvironment. Obese C57Bl/6J mice were transplanted with GFP+ murine preB cell ALL (“8093”) cells and then treated with vincristine (0.5 mg/kg/week × 3 weeks). At the time of relapse, we found that GFP+ leukemia cells persisted in the fat pads of the mice. We then developed an in vitro co-culture system in which human or murine leukemia cells were cultured together with adipocytes (differentiated 3T3-L1s). Undifferentiated 3T3-L1 cells, which are fibroblastic in nature, were used as a control. In this model, adipocytes severely diminished the anti-leukemic effect of all chemotherapeutics tested against murine 8093 cells, including vincristine, dexamethasone, nilotinib, daunorubicin, and L-asparaginase. Adipocytes also protected murine T-cell ALL and human SD-1, RCH-ACV, and BV173 cells from vincristine and daunorubicin. Adipocyte protection of leukemia cells occurred independent of cell contact. Further experiments demonstrated that media conditioned by adipocytes was able to protect 8093 cells from a 3-day exposure to 25 nM dexamethasone (viable cells were at 40±12% of their plated value in regular media, 66±17% in fibroblast-conditioned media, and 109±24% in adipocyte-conditioned media, p<0.05). Surprisingly, adipocyte-conditioned media did not protect leukemia cells from daunorubicin. However, media conditioned by the presence of both adipocytes and leukemia cells simultaneously conferred a high degree of resistance to the leukemia cells (n=3, p<0.05 compared to all other media types). In summary, adipose tissue is a reservoir for relapsed leukemia cells in vivo. Adipocytes engender protection from multiple chemotherapies in murine and human leukemia cell lines. Adipocytes secrete factor(s) that confer dexamethasone and daunorubicin resistance to leukemia cells, though for the latter drug it appears that a two-way communication between leukemia and adipocytes may be necessary for this protection. Figure Figure

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Small Molecule Inhibition of Cdc7, a Key Cell Cycle Regulator and Novel Therapeutic Target, Successfully Inhibits Leukemia Cell Growth in Vitro and in Vivo

Blood ◽

10.1182/blood.v112.11.2668.2668 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2668-2668

Author(s):

Mark G. Frattini ◽

David Shum ◽

Kristen M O’Dwyer ◽

Renier J. Brentjens ◽

Ray Yeh ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Small Molecule ◽

Leukemia Cell ◽

Standard Cell ◽

Cdc7 Kinase ◽

Kinase Inhibitory Activity ◽

Novel Therapeutic

Abstract Cdc7 is a heterodimeric serine/threonine protein kinase that is a key regulator in the process of initiation of DNA replication and the G1 to S phase transition. Both the kinase and its known substrates are over-expressed in the majority of human cancers. As a result of the recent progress in the areas of pharmacogenetics and high throughput screening technology, identifying specific small molecule inhibitors of cell cycle regulated protein kinases has provided a means not only to study these signal transduction pathways but also to identify potential novel therapeutic agents. To this end, we have developed an assay for Cdc7 kinase inhibitory activity using a highthroughput screening (HTS) approach, screening over 250,000 natural and synthetic small molecules. As a result, we have identified and confirmed seventeen compounds, representing nine different chemical scaffolds, with Cdc7 kinase inhibitory activity. Based on potency, we selected the lead compound (CKI-7) which was further characterized using kinase profiling, microarray experiments, and standard cell based cytotoxicity assays. These latter studies demonstrated that CKI-7 induced cytotoxicity of established leukemia and lymphoma cell lines in culture with inhibitory concentrations (IC50s) in the low nanomolar range. Significantly, CKI-7 likewise induced cytotoxicity of MDR1 overexpressing cell lines with similar IC50s, demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. We additonally demonstrate that CKI-7 induces cytotoxicity of patient-derived primary acute leukemia tumor cells (both chemotherapy naïve and relapsed/refractory samples) in vitro at similarly low nanomolar concentrations. In vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilzing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Standard cell cycle synchronization studies established that exposure to CKI-7 results in cell cycle dependent caspase 3 activation and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a substrate biomarker assay. In conclusion, our data confirm that Cdc7 is a new promising target for cancer therapy, and that CKI-7, a selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent.

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Small-Molecule Inhibition of BRD4 Is a Novel Promising Approach to Therapeutically Target Leukemia Stem Cells in AML,

Blood ◽

10.1182/blood.v118.21.3484.3484 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3484-3484 ◽

Cited By ~ 1

Author(s):

Harald Herrmann ◽

Katharina Blatt ◽

Junwei Shi ◽

Amy R. Rappaport ◽

Karoline V. Gleixner ◽

...

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Mouse Models ◽

Small Molecule ◽

Conflicts Of Interest ◽

Leukemia Stem Cells ◽

Clinical Value ◽

In Vivo Evaluation ◽

Small Molecule Inhibition

Abstract Abstract 3484 Acute myeloid leukemia (AML) is a stem cell-derived malignancy characterized by uncontrolled proliferation and accumulation of myeloblasts in hematopoietic tissues. The clinical course and prognosis in AML vary depending on deregulated genes, cell type(s) involved, and the biological properties of the clone. In most variants of AML, the complexity and heterogeneity of oncogenomes pose a challenge for the development of effective targeted therapeutics. However, diverse genetic aberrations in AML typically converge functionally to dysregulate the same cellular core processes. One key event is the corruption of myeloid cell-fate programs resulting in the generation of aberrantly self-renewing leukemia stem cells (LSC), which maintain and propagate the disease and are often resistant to conventional chemotherapy. Hence, strategies aimed at terminating aberrant self-renewal and eradicating LSC are considered as key for the development of more effective AML therapies. In an effort to systematically probe genes involved in chromatin regulation as potential therapeutic targets, we recently employed an unbiased screening approach combining AML mouse models and new in-vivo RNAi technologies, through which we identified the epigenetic ‘reader' BRD4 as new candidate drug target in AML (Zuber et al., Nature, in press). Inhibition of BRD4 using RNAi or a new small-molecule inhibitor (JQ1) blocking BRD4 binding to acetylated histones, showed profound antileukemic effects in AML mouse models, in all human AML cell lines tested (n=8) as well as in primary AML cells. In all models tested, BRD4 suppression was found to trigger apoptosis as well as terminal myeloid differentiation, and potently suppressed expression programs previously associated with LSC. As one key target, we observed a dramatic transcriptional repression of MYC, which recently has been discussed as core component of an LSC associated transcriptional module. To further evaluate suppression of BRD4 as a potential therapeutic approach to eradicate LSC in human AML, we analyzed the effects of JQ1 in primary AML cells obtained from 17 patients with freshly diagnosed or relapsed/refractory AML (females, n=5, males, n=12, median age: 54 years; range: 21–80 years). In unfractionated primary AML cells, submicromolar doses of JQ1 were found to induce major growth-inhibitory effects (IC50 between 0.05 and 0.5 μM) in a broad spectrum of AML subtypes. No differences in IC50 values were seen when comparing drug effects in AML cells kept in the presence or absence of growth-stimulating cytokines (G-CSF, IL-3, SCF). In addition, JQ1 treatment effectively triggered apoptosis in all patients tested, with similar anti-leukemic activities observed in newly diagnosed pts and refractory/relapsed AML. To further evaluate the clinical value of BRD4 as a clinically relevant target in AML, we analyzed the effect of JQ1 on AML LSC. In these experiments, JQ1 effectively induced apoptosis in CD34+/CD38+ progenitor cells as well as in CD34+/CD38− AML stem cells in all donors examined as evidenced by combined surface/Annexin-V staining. Furthermore, JQ1 was found to induce morphologic signs of maturation in 6 of 7 patients examined, thereby confirming our previous data obtained in mouse AML cells. Finally, we were able to show that JQ1 synergizes with Ara-C in inducing growth inhibition in HL60 cells and KG-1 cells. In summary, our data show that small-molecule inhibition of BRD4 has strong anti-leukemic effects in a broad range of AML subtypes. Furthermore, our results support the notion that JQ1's ability to suppress LSC specific transcriptional modules may translate into a therapeutic entry point for eradicating LSC in primary AML. While a more extensive in vivo evaluation of these effects, as well as the development of pharmacologically improved compounds will be required, all existing data unambiguously highlight small-molecule inhibition of BRD4 as a new promising concept in AML therapy. Disclosures: No relevant conflicts of interest to declare.

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