RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

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Clathrin Assembly Protein CALM Is Necessary for Leukemia Cell Proliferation and Erythroid Development Via Regulating Transferrin Receptor Endocytosis

Blood ◽

10.1182/blood.v118.21.244.244 ◽

2011 ◽

Vol 118 (21) ◽

pp. 244-244

Author(s):

Yuichi Ishikawa ◽

Manami Maeda ◽

Min Li ◽

Sung-Uk Lee ◽

Julie Teruya Feldstein ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemia Cell ◽

Leukemia Cells ◽

Leukemia Cell Lines ◽

Erythroid Development

Abstract Abstract 244 Clathrin assembly lymphoid myeloid leukemia (CALM) protein is implicated in clathrin dependent endocytosis (CDE) and the CALM gene is the target of the t(10;11)(p13;q14-21) CALM/AF10 translocation, which is observed in multiple types of acute leukemia. Although the translocation generally dictates poor prognosis, the molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. To determine the role of CALM and CDE in normal hematopoiesis and leukemogenesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/FMx1Cre+) Calm knockout (KO) mutants. Furthermore, we determined the impact of Calm loss on leukemia cell growth in vitro and in vivo employing a series of leukemia cell lines and leukemia mouse models. Hematopoietic-specific Calm knockout mice (CalmF/FMx1Cre+) exhibited a hypocromatic anemia with increased serum iron levels. We observed significant reduction in mature erythroblasts/erythrocytes (TER119+CD71-) with concomitant increase in immature erythroblasts (TER119+CD71+) in the spleen of CalmF/FMx1Cre+ mice. The frequencies of erythroblasts in S phase were lower and the proportions of apoptotic (cleaved PARP positive) erythroblasts were increased in CalmF/FMx1Cre+ mice. Surface transferrin receptor 1 (Tfr1, CD71) levels were significantly up-regulated in Calm-deficient hematopoietic progenitors, and uptake of Alexa647-conjugated transferrin was abrogated in Calm-deficient erythroblasts, revealed by immunofluorescence analysis. Freez-etch electron microscopy analysis showed a defective clathrin coated vesicle (CCV) formation in Calm-deficient erythroblasts, indicating that Calm is indispensable for iron-bound transferrin internalization by regulating CCV formation, thereby critical for erythroid differentiation and hemoglobinization. CALM was highly expressed in leukemia/lymphoma cell lines and primary acute myeloid leukemia samples, although its expression was limited to erythroblasts in normal hematopoietic lineage cells. Treatment of leukemia cell lines with Desferoxamine (DFO), an iron chelator, led to a significant increase in Calm mRNA levels, suggesting that Calm expression is regulated by intracellular iron levels. Since highly proliferative leukemia cells demand iron as a cofactor for ribonucleotide reductase (RNR), we hypothesized that Calm is required for leukemia cell proliferation by regulating iron-bound transferrin internalization. To determine the effect of Calm inactivation in leukemia cells, we transduced a series of leukemia cell lines with a lentivirus-based ShRNA vector (pLKO-GFP), which allowed shRNA-expressing cells to be traced by green fluorescent protein (GFP). Calm shRNA transduced cells, but not cells transduced with scrambled shRNA, showed a proliferative disadvantage compared to non-transduced cells. To determine the effect of Calm deletion in leukemia cells in vivo, the CALM/AF10 oncogene was retrovirally transduced into either wild type (WT) or CalmF/FMx1Cre+ bone marrow (BM) cells and the cells were subsequently transferred to lethally-irradiated recipient mice. The Calm gene was deleted in donor cells via pIpC injections one month after transplant (before leukemia development) and survival curves generated. The recipients transplanted with the BM cells from CalmF/FMx1Cre+ mice showed a significantly delayed onset of leukemia and longer survivals compared to control (p=0.001), indicating that Calm is necessary for the development of CALM/AF10-induced leukemia. We next assessed whether Calm is required for the “maintenance” of leukemia in vivo. Leukemia cells were harvested from the primary recipients transplanted with the CALM/AF10-transduced CalmF/FMx1Cre+ BM cells (in which the endogenous Calm genes were intact) and transferred to the secondary recipients. The leukemic secondary recipient mice were then injected with pIpC and survival curves generated. Calm inactivation significantly delayed leukemia progression by blocking leukemia cell proliferation. Taken together, our data indicate that Calm is essential for erythroid development and leukemia cell proliferation by regulating TFR1 internalization. Since Calm inactivation significantly blocked the leukemia cell proliferation in vitro and in vivo, our findings may provide new therapeutic strategies for acute myeloid leukemia. Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

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In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.2605.2605 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2605-2605

Author(s):

Lars Bullinger ◽

Konstanze Dohner ◽

Richard F. Schlenk ◽

Frank G. Rucker ◽

Jonathan R. Pollack ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Serum Levels ◽

Leukemia Cell Lines ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

Abstract Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

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The 3-Drug Combination Treatment ART838, ABT199 Plus Sorafenib Is Effective Against AML Xenografts, Possibly Via Cooperative Targeting of MCL1

Blood ◽

10.1182/blood-2018-99-113040 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4044-4044

Author(s):

Blake S Moses ◽

Jennifer Fox ◽

Xiaochun Chen ◽

Samantha McCullough ◽

Sang Ngoc Tran ◽

...

Keyword(s):

Acute Leukemia ◽

Board Of Directors ◽

Cell Lines ◽

Drug Combination ◽

Research Funding ◽

Leukemia Cell ◽

Advisory Committees ◽

Leukemia Cell Lines ◽

Equity Ownership

Abstract Antimalarial artemisinins have broad antineoplastic activity in vitro, are well tolerated and inexpensive, and can be parenterally or orally administered in humans. Artemisinin-derived trioxane diphenylphosphate dimer 838 (ART838; a potent artemisinin-derivative) inhibited acute leukemia growth in vivo and in vitro, at doses where normal human CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected (Fox et al, Oncotarget 2016, PMID: 26771236). In our focused drug combination screen for drugs that synergize with ART838, the only BCL2 inhibitors in the screen library of 111 emerging antineoplastic compounds, navitoclax (ABT737) and venetoclax (ABT199; FDA-approved), were identified as 2 of the top 3 candidates. Synergies between ART838 and BCL2 inhibitors were validated in multiple acute leukemia cell lines and primary cases. This ART838-BCL2 inhibitor synergy may be due to reduced levels of MCL1 protein that we and others have observed in multiple acute leukemia cell lines and primary cases treated with artemisinins (Budhraja et al, Clin Cancer Res 2017, PMID: 28974549). Treatment of acute leukemia xenografts with the ART838 plus ABT199 combination reduced leukemia growth rates and prolonged survivals, compared to vehicle or either ART838 or ABT199 alone. To add to the efficacy of this ART838 plus ABT199 treatment regimen, we sought to rationally add a third low-toxicity active antileukemic agent. Sorafenib (SOR; FDA-approved) inhibits multiple kinases which may mediate its antileukemic activity, with the importance of the targets varying from case to case; e.g. FLT3 is an important target in many AMLs. In addition, several reports have found that SOR reduces MCL1 protein stability and translation through inhibition of the ERK and PI3K pathways (Wang et al, Clin Cancer Res 2016, PMID: 26459180; Huber et al, Leukemia 2011, PMID: 21293487). In all acute leukemia cell lines tested, we observed large reductions in MCL1 protein levels with SOR treatment, which may further rationalize the addition of SOR to our ART838 plus ABT199 antileukemic regimen. We had previously observed strong in vitro synergy between ART838 and SOR (PMID: 26771236). Treatment of acute leukemia xenografts with the ART838 plus SOR combination reduced leukemia xenograft growth rates and prolonged survivals, compared to single drugs. Mice bearing luciferase-labelled acute leukemia xenografts were treated (PO daily x5) with single drug or 2-drug or 3-drug combinations of ART838, ABT199, and SOR, each at their individual maximally tolerated doses. Treatment with this 3-drug combination caused rapid regression of luciferase-labelled MV4;11 AML xenografts (Fig 1A). The 5-day treatment cycles were repeated every other week, and mice receiving this 3-drug combination survived >4 times longer than vehicle-treated mice (Fig 1B). Mouse body weights were stable during treatment. Although myelosuppression is the human clinical dose-limiting toxicity of each of these 3 drugs, mouse blood cell counts during 3-drug combination treatment were in the normal range. Treatment of a luciferase-labelled primary AML leukemia xenograft with this 3-drug combination reduced leukemia growth more than the single drugs or 2-drug combinations (Fig 1C). Assessment of efficacy and pharmacokinetics-pharmacodynamics against diverse acute leukemia xenografts will test this combination of ART838, ABT199 plus SOR as a rational low-toxicity drug triad for treatment of acute leukemias and potentially other cancers. Disclosures Fox: Intrexon Corporation: Employment. Tyner:Genentech: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Array: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aptose: Research Funding. Civin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 3DBioWorks Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria.

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A Novel N-Oxide Drug, AQ4N, Has In Vitro Activity in Lymphoma and Leukemia Cell Lines and Selectively Targets Lymphocytes and Lymphatic Tissues in Vivo.

Blood ◽

10.1182/blood.v104.11.2498.2498 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2498-2498

Author(s):

Jeffrey L. Cleland ◽

Alvin Wong ◽

Susan E. Alters ◽

Peter A. Harris ◽

Chris R. Dunk ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Lymphoid Tissues ◽

Human Leukemia ◽

Repeat Dose ◽

Cynomolgus Monkeys ◽

Leukemia Cell Lines ◽

Human Lymphoma

Abstract An ideal treatment for lymphoma and leukemia is the use of highly selective compounds to eliminate diseased cells with minimal systemic toxicity to normal tissues (cf. imatinib mesylate; Gleevec). AQ4N (1,4 bis[[2-(dimethylamino)ethylamino}-5,8-hydroxyanthracene-9,10-dione bis N-oxide) is designed to have little or no toxicity until selectively activated by bioreduction in hypoxic cells to AQ4 (reduced AQ4N), a highly potent DNA topoisomerase II inhibitor. In a series of studies, AQ4 has been shown to have potent cytotoxicity on lymphoma and leukemia cell lines in vitro and AQ4N has selective activity in lymphatic tissues in vivo. The IC50 of AQ4, was 0.63, 12.0, 90.5 and 150 nM in Namalwa, Daudi, Ramos, and Raji human lymphoma cell lines and 1.0, 6.0, and 20 nM in HL-60, KG1a and K562 human leukemia cell lines. On several of the tumor lines the activity of AQ4 was more potent than doxorubicin (i.e. IC50 for Dox was 20.3 nM on Namalwa). AQ4N also had anti-proliferative activity at μM levels indicating a potential mechanism for activation by these cell lines. In repeat dose toxicology studies of AQ4N in pigmented rats and cynomolgus monkeys, the maximum tolerated doses (MTD; rats: 20 mg/kg/wk x 6; monkeys 6 mg/kg/wk x 6) resulted in lymphoid tissue atrophy. A decrease in lymphocyte levels and atrophy of the spleen, thymus, and mandibular and mesenteric lymph nodes were observed at terminal sacrifice of the animals. In contrast, there was an absence of myelosuppression and only mild neutropenia and minor bone marrow atrophy at the MTD. Administration of radiolabeled AQ4N (14C-benzene) to pigmented rats and cynomolgus monkeys indicated persistence of AQ4N radioactivity in lymphoid tissues for several weeks after a single dose (rats: 20 mg/kg (130–140 μCi/kg); monkeys: 10 mg/kg (135 μCi/kg)). For example, in rats the half-life of radioactive AQ4N in the spleen was 538 hrs with 0.9 μg AQ4N/g tissue (spleen) remaining one week after dosing. Monkeys demonstrated a similar effect with 76.5–86.8 μg AQ4N/g tissue observed in the spleen one week after treatment. Other tissues contained significantly less radioactive AQ4N with the exception of the liver (67.9–78.6 μg AQ4N/g tissue) and adrenal cortex (78.7–86.6 μg AQ4N/g tissue). While some hypertrophy and eosinophila was observed in the adrenal glands, liver toxicity was not observed at the MTD in the repeat dose cynomolgus monkey toxicology study. Overall, these initial findings indicate that AQ4N is active in vitro against human lymphoma and leukemia cell lines and selectively targets lymphoid tissues in vivo suggesting the potential benefit of AQ4N in the treatment of lymphoproliferative diseases.

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Characterization of Double BCR-ABL–positive Cell Lines Established from a Patient with Blasic Crisis of Chronic Myeloid Leukemia Who Showed Clinical Resistant to Imatinib

Blood ◽

10.1182/blood.v112.11.4244.4244 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4244-4244

Author(s):

Tsuyoshi Nakamaki ◽

Norimichi Hattori ◽

Hidetoshi Nakashima ◽

Takashi Maeda ◽

Hirotsugu Ariizumi ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Kinase Domain ◽

Abl Kinase ◽

Leukemia Cell Lines ◽

Clinical Resistance

Abstract Pervious in vitro studies have shown that molecular alterations of BCR-ABL-positive leukemia cells such as amplification of BCR-ABL gene and/or mutation(s) of abl kinase domain cause resistant to imatinib. However recent study showed that alterations of imatinib bioavailability might be a important factor to cause clinical resistant in BCR-ABL-positive leukemia patients, showing a differences between in vivo and in vitro sensitivity to imatinib of BCR-ABL-positive cells. To analyze mechanism(s) of clinical resistance to imatinib and to overcome the resistance, we have sequentially established and characterized two leukemia cell lines from a patient with myeloid blastic crisis of chronic myeloid leukemia (CML) who showed progressively resistant to imatinib. Case report and establishment of cell lines: a 59-years-old women developed blastic crisis preceded by four years of chronic phase of CML. Increased blasts in crisis was positive for CD13, 33 and showed double Ph-chromosome in addition to complexed chromosomal alterations such as, add(3)(p13), add(3)(q11), add(5)(q11), der(19)(3;19) (p21;q13). After repeated courses of combination chemotherapy including, 600mg of imatinib was administered orally in combination with chemotherapeutic drugs. For a brief period Imatinib showed clinical effects and slowed the increase of BCR-ABL-positive cells, however myeloblast progressively increased in peripheral blood in spite of daily administration of imatinib and she died four months treatment with imatinib. Two myeloid leukemia cell lines, NS-1 and NS-2 were established, after obtaining informed consent, from peripheral blood at day 65 and day 95 after initiation of imatinib administration, respectively. Cell surface phenotype and karyotype of these cell lines were identical to original blasts. NS-1 and NS-2 cell lines were characterized compared with BCR/ABL-positive K562 erythroleukemia cell line as a control Quantitative analysis by real-time polymerase chain reaction showed that copy number of BCR-ABL transcript were 2.2 × 105 and 1.6 × 10 5/μg RNA in NS-1 and NS-2 respectively, showing slightly lower than those (5.8 × 105) in K562 cell line. Although nucleotide sequence analysis showed that a point mutation in abl kinase domain resulted in amino acid substitution pro310ser in NS-1 cell line, no additional mutation was found in NS-2 cell line. Western blot analysis showed levels of both 210 KD BCR-ABL protein and BCR-ABL phosphorylation were similar in NS-1, NS-2 and K562 cells. Although two hours incubation with 10 mM imatinibin vitro did not show any detectable difference in levels of phosphorylation of BCR-ABL protein between NS-1 and NS-2 cell lines, sensitivity to imatinib measured by MTT assay showed that IC50 was 0.1 mM, 0.5 mM and 1.0mMin NS-1, NS-2 and K562 cell lines respectively. The measured IC50 of both NH-1 and NH-2 cell lines were much lower than reported plasma concentrations achieved by oral administration of 600 mg of imatinib (above 10 μM). The present results suggest difference between in vivo and in vitro sensitivity to imatinib indicate that alteration of bioavailability of imatinib possibly involved in clinical resistance to this drug, accumulations of BCR-ABL gene amplification and/or mutation are not necessarily a major reason of progressive clinical resistance to imatinib in BCR-ABL positive leukemia.

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The Novel Sulfonamidebenzamide ML291 Activates Apoptotic UPR Signaling in Pediatric Leukemia

Blood ◽

10.1182/blood.v128.22.3523.3523 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3523-3523

Author(s):

Danielle Garshott ◽

Nicole Melong ◽

Tania T. Sarker ◽

Yue Xi ◽

Amy Brownell ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Leukemic Cell ◽

K562 Cells ◽

Leukemia Cells ◽

Wild Type ◽

Leukemia Cell Lines ◽

Proliferation Assays

Abstract Background: Acute leukemias are the most common cancers in childhood. Despite multi-agent chemotherapy protocols and the introduction of novel molecularly targeted therapies which have resulted in improved survival over the last few decades, relapsed acute lymphoblastic leukemia remains the second most common pediatric cancer diagnosis. In addition, morbidities from current chemotherapy regimens are unacceptably high. Abundant evidence point to a major role for mediators of the unfolded protein response (UPR) in normal and leukemic white blood cell biology. We have demonstrated that activation of the UPR is a productive approach to inhibit the proliferation of solid tumor cell lines in vitro and to reducing xenograft burden in vivo. The UPR consists of genetically distinct mechanisms that serve to clear misfolded proteins from the endoplasmic reticulum (ER) and enhance protein folding, or induce apoptosis if the initiating stress is prolonged or robust. ML291 is a novel UPR-inducing sulfonamidebenzamide, identified through cell-based high throughput screening and iterative SAR-guided chemical synthesis, that overwhelms the adaptive capacity of the UPR and induces apoptosis in a variety of solid cancer models. Objective: To determine the ability of ML291 to activate the UPR and induce apoptosis in a panel of leukemia cell lines, and to use CHOP-null K562 cells to elucidate the relative contribution of the UPR. We hypothesized that ML291 might activate the PERK/eIF2a/CHOP (apoptotic) arm of the UPR and reduce leukemic cell burden in vitro and in vivo. Methods: MTT and luciferase-based proliferation assays, flow cytometry and RT-qPCR were used to evaluate cell growth, UPR activation and apoptosis in a panel of leukemia cell lines that included AML, ALL and CML in cells exposed to ML291. CRISPR-Cas9 genome editing was used to delete CHOP in K562 (human myeloid leukemia) cells. Deletion was validated by immunoblot analysis and these cells were subjected to the same proliferation and gene analyses described above. The in vivo response to ML291 therapy was evaluated in an established zebrafish xenograft assay (Corkery et al. BJH 2011) in which embryos were xenotransplanted with wild type or CHOP knockdown K562 cells and embryos bathed in ML291. Results: Immunoblot and RT-qPCR analysis revealed an accumulation of proteins and increased gene expression for downstream UPR genes, including CHOP, GRP78/BiP, GADD34 and XBP1 in leukemia cells following ML291 treatment, indicating the activation of the UPR. Increased expression of the apoptotic genes, NOXA, PUMA and DR5 was also observed post-treatment with ML291; and dose response proliferation assays performed after 24 hours revealed IC50 concentrations of 1 - 30µM across cell lines. CHOP deleted K562 cells were protected from cell death when cultured with increasing concentrations of ML291, and were significantly less able to translocate phosphatidylserine across the cell membrane and activate the caspase cascade. When zebrafish embryos xenotransplanted with K562-wild type or -CHOP-null cells were bathed in water containing 5mM ML291 for three days, there was a significant reduction in leukemia cell burden exclusively in theK562 wild type xenografts. Conclusion: Collectively these data indicate that intact PERK/eIF2a/CHOP signaling is required for efficient leukemic cell apoptosis in response to ML291 in vitro and in vivo, and support the hypothesis that small molecule enforcement of the UPR might be a productive therapeutic approach in leukemia. Disclosures No relevant conflicts of interest to declare.

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Development of Astatine-211 (211At)-Based Anti-CD123 Radioimmunotherapy for Acute Leukemias and Other CD123+ Hematologic Malignancies

Blood ◽

10.1182/blood-2021-152105 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3341-3341

Author(s):

George S. Laszlo ◽

Johnnie J. Orozco ◽

Allie R. Kehret ◽

Margaret C. Lunn ◽

Donald K. Hamlin ◽

...

Keyword(s):

Acute Leukemia ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Leukemia Cell ◽

Hematologic Malignancies ◽

Advisory Committees ◽

Leukemia Cell Lines ◽

Efficacy Studies

Abstract Background: Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia. Of current interest are alpha-particle emitting radionuclides as they deliver a very large amount of radiation over just a few cell diameters, enabling efficient and selective target cell kill. So far, alpha-emitters including astatine-211 (211At) have been primarily explored with monoclonal antibodies (mAbs) targeting CD45 or CD33 but their broad display on non-malignant target-expressing cells can lead to marked "on-target, off tumor cell" toxicities. To overcome this limitation, we developed a novel form of 211At-based RIT targeting CD123. CD123 is displayed widely on acute leukemia cells, including underlying leukemic stem cells, but is expressed only on a discrete subset of normal hematopoietic cells and is virtually absent on non-blood cells. Methods: We immunized BALB/c mice with peptides consisting of the extracellular domain of human CD123 to generate anti-CD123 mAbs. Flow cytometry-based assays with human acute leukemia cell lines were used to characterize binding of hybridoma supernatants and mAbs to CD123. mAbs were conjugated with isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10), a boron cage molecule for subsequent astatination, and were then labeled with 211At. In vivo leukemia cell targeting ("biodistribution") and efficacy studies were conducted in immunodeficient NOD-Rag1 null IL2rɣ null/J (NRG) mice xenografted with MOLM-13 cells, a CD123+ human acute myeloid leukemia cell line. Results: Based on initial hybridoma screening studies, we selected 4 mAbs (10C4, 5G4, 11F11, and 1H8) for further characterization. Phenotyping studies with CD123+ and CD123- human acute leukemia cell lines (including CD123+ cell lines in which CD123 was deleted via CRISPR/Cas9) confirmed specific binding of all mAbs to human CD123 (binding intensity: 10C4>5G4=11F11=1H8), with 10C4 yielding a higher median fluorescence intensity than the widely used commercial anti-CD123 mAb clones, 7G3 and 6H6 (Figure 1). In vitro internalization with a panel of human acute leukemia cell lines studies demonstrated uptake of all mAbs by CD123+ target cells with a kinetic slower than that for anti-CD33 antibodies (typically, 30-50% of the anti-CD123 mAb internalized over 2-4 hours). All 4 anti-CD123 mAbs could be conjugated to B10 and subsequently labeled with 211At. Unlike a non-binding 211At-labeled control mAb, 211At-labeled anti-CD123 mAbs showed uptake at MOLM-13 flank tumors in NRG mice carrying MOLM-13 xenografts. After additional leukemia cell targeting studies to optimize the dosing of 10C4, we conducted proof-of-concept efficacy studies in NRG mice injected intravenously with luciferase-transduced MOLM-13 cells (disseminated leukemia model). Animals were either untreated or treated with 10 µCi, 20 µCi, or 40 µCi of 211At-labeled 10C4-B10 mAb (9-11 animals/group). This was followed by the infusion of bone marrow cells from donor mice as stem cell support 3 days later. As shown in Figure 2 and Figure 3, 211At-10C4-B10 led to a dose dependent decrease in tumor burden. Further, the treatment significantly prolonged survival compared to untreated animals (median survival: 49 days [40 µCi of 211At] vs. 31 days [10 µCi of 211At] vs. 21 days [Ctrl]; P<0.0001 for Ctrl vs. 10 µCi, P<0.004 for 10 µCi vs. 40 µCi), demonstrating potent in vivo anti-leukemia efficacy of a single dose of 211At-CD123 RIT. Conclusion: Our data support the further development of 211At-CD123 RIT for the treatment of patients with acute leukemia and other CD123+ hematologic malignancies. Figure 1 Figure 1. Disclosures Green: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; JANSSEN Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

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The Activity of Combined Topoisomerase Inhibitors Is Augmented by Octreotide in the Treatment of Lymphoid Leukemias.

Blood ◽

10.1182/blood.v106.11.2475.2475 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2475-2475

Author(s):

Dimitrios T.P. Trafalis ◽

Konstantinos Lilakos ◽

Daphne Koumbi ◽

Panayiotis Dalezis ◽

Maria K. Angelopoulou ◽

...

Keyword(s):

Cell Lines ◽

Antitumour Activity ◽

Leukemia Cell ◽

Mrna Levels ◽

In Vitro Growth ◽

Antagonistic Effects ◽

Leukemia Cell Lines ◽

Topo Ii

Abstract Clinical protocols combining a topoisomerase I (topo I) and a topoisomerase IIalpha (topo II inhibitor) have shown major responses against several tumours including acute leukemia and myelodysplastic syndromes. However, combinations with topoisomerase targeting drugs should be considered with caution because antagonistic effects have been observed when administering camptothecin or topotecan (topo I inhibitors) with doxorubicin (topo II inhibitor). Octreotide (OCT) is an eight amino-acid peptide, which attains its biological effects on target cells by binding preferentially to sst2 and, to a lesser extent, to sst3 and sst5 somatostatin receptors (SS-Rs). The presence of SS-Rs in human lymphoid leukemia cell lines, in malignant lymphomas and in lymphoproliferative diseases is clearly detected. We studied the in vitro effect of combinations of OCT with doxorubicin (DOX) and topotecan (TP) on cell growth and viability in four human lymphoblastic leukemia cell lines (CCRF-CEM, RPMI-8226, JURKAT, MOLT-4) and on the Topo I, IIalpha and sst2 expression in JURKAT, MOLT-4 leukemia cell lines, as well as the in vivo effect on rodent P388 lymphocytic and L1210 lymphoid leukemias. In vitro growth inhibition and cytotoxicity were evaluated with the MTT colormetric metabolic assay. Topo I, IIalpha and sst2 mRNAs were detected with RT-PCR and the quantification of the electrophoresed specific PCR products was accomplished with Molecular Imager FX. In vivo antitumour activity was estimated by the % survival ratio of treated to untreated (control) mice and the ratio of 70-day tumour free survivors (cures). The in vitro growth inhibition and cytotoxicity that were induced by the DOX and TP combinations were neither synergistic nor additive and were similar to the activity of DOX alone. However, with the addition of OCT to the DOX and TP combinations a significant (p<0.001) synergistic effect was resulted in all tested cell lines. Treatment of cell lines with DOX produced almost a total consumption of Topo I and IIalpha mRNAs and with TP induced increase of Topo IIalpha mRNA levels (0.5–1.5 folds). OCT is clearly upregulates the Topo IIalpha expression (>2.5 folds) and restores Topo I mRNA production in cells treated with DOX. The sst2 mRNA levels were not affected in any case. In vivo antitumour activity of DOX and TP combinations was neither synergistic nor additive and it was similar to the activity of DOX alone. The addition of OCT to DOX and TP combinations produced an important synergistic antitumour effect increasing significantly survival time and cures (p<0.01) in both P388 and L1210 leukemias. Our data indicate that the antagonistic effects of Topo I and Topo II inhibitors may be due to effects on the regulation of topoisomerases expression. OCT significantly enhances the antileukemic activity of combinations with such important anticancer drugs, upregulating Topo I and IIalpha expression.

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

Blood ◽

10.1182/blood.v122.21.3962.3962 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3962-3962

Author(s):

Laura M Bystrom ◽

Hongliang Zong ◽

Hsiao-Ting Hsu ◽

Neng Yang ◽

Noa Greenberg ◽

...

Keyword(s):

Cell Lines ◽

Iron Metabolism ◽

Leukemia Cell ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Survival Pathways ◽

Leukemia Cell Lines ◽

Acute Myelogenous

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

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DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines

Blood ◽

10.1182/blood-2001-11-0032 ◽

2002 ◽

Vol 100 (12) ◽

pp. 3968-3974 ◽

Cited By ~ 55

Author(s):

Hiroshi Nishihara ◽

Masae Maeda ◽

Atsushi Oda ◽

Masumi Tsuda ◽

Hirofumi Sawa ◽

...

Keyword(s):

Cell Lines ◽

Cell Attachment ◽

Leukemia Cell ◽

Jurkat Cells ◽

Human Leukemia ◽

Lymphocyte Migration ◽

Leukemia Cell Lines ◽

Src Homology

The CDM (ced-5 of Caenorhabditis elegans,DOCK180 [downstream of Crkwith molecular weight of 180 kDa] of humans, andmyoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor moleculec-CT10–related kinase II (c–Crk-II) in adherent cells. Recently, hematopoietic cell–specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c–Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines.

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