Profiling of 24 Standard of Care Drugs in a Panel of 20 Human Hematological Cell Lines Using Xenograft-Derived Three-Dimensional (3D) Cultures Ex Vivo and In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4995-4995
Author(s):  
Armin Maier ◽  
Monika Engelhardt ◽  
Heinz-Herbert Fiebig ◽  
Julia Schüler
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Hematological Malignancies ◽  
Ex Vivo ◽  
Clonogenic Assay ◽  
Three Dimensional ◽  
Standard Of Care ◽  
Tumor Xenografts

Abstract Abstract 4995 Introduction: Leukemia and lymphoma account for a notable proportion of cancers worldwide. The heterogeneity and biological characteristics of hematological malignancies induce unique therapeutic challenges. It is well known that pluripotent as compared to differentiated cells possess the potential for anchorage independent growth in semisolid medium. This can be monitored via clonogenic or colony formation assays, in which cells grow in vitro in a three-dimensional (3D) manner without adherence to plastic culture material support. These assays can be utilized to evaluate growth and drug sensitivity of tumor stem and progenitor cells (Fiebig HH et al. Eur J Cancer 40:802, 2004). In addition, these 3D cell culture assays often mimic the in vivo scenario better than 2D cell culture assays with adherent tumor cells. Material and methods: For our ex vivo anti-tumor efficacy profiling using clonogenic assays, we established a panel of 20 hematological cell lines comprising different entities like acute lymphoblastic leukemia (ALL, 4 cell lines), acute myeloid leukemia (AML, 6 cell lines), chronic myeloid leukemia (CML, 5 cell lines), Hodgkin- (1 cell line) and non-Hodgkin-lymphoma (NHL, 3 cell lines), as well as multiple myeloma (MM, 3 cell lines). Tumor cells were injected into the flanks of NOD/SCID mice in order to obtain subcutaneous tumor xenografts, which were kept at low passages (n <3). These xenografts served as starting material either to prepare single cell suspensions for ex vivo analysis, or to carry out in vivo efficacy tests using either subcutaneous or disseminated growing tumor xenografts. Results: Twenty-four standard of care agents were tested in terms of their ex vivo chemosensitivity (e.g. cytarabine, cyclophosphamide, dexamethasone, doxorubicin, etoposide, melphalan, prednisolone, vincristine), including selected targeted drugs also (e.g. bortezomib, imatinib, nilotinib, sorafenib). The drugs showed diverse patterns of selectivity and potency: vincristine, doxorubicin and cytarabine, but also the proteasome inhibitor bortezomib exhibited pronounced activity with IC50 values in the nanomolar range (mean IC50 = 1 – 100nM), not only in their respective clinical application, but also in various other tumor entities, such as in ALL and AML with use of bortezomib. Differential activity was determined e.g. for prednisolone and dexamethasone, which were active in a micromolar range (mean IC50 = 22 – 58μM) in the ALL cell lines CCRF-CEM and MOLT-4, AML cell lines NOMO-1, NHL DAUDI and U-937, as well as the MM cell line IM-9. All-trans-retinoic acid (mean IC50 = 1.3μM) as well as interferon-gamma-1b (mean IC50 = 0.43 μM) showed specific activity patterns with pronounced growth inhibition in AML (3/6 tested AML cell lines: KG-1, NOMO-1, OCI-AML2), but also in CML (1/5 tested CML cell lines: EM-2) and MM (1/3 tested MM cell lines: L-363). The strong correlation of both tyrosine kinase inhibitors imatinib and nilotinib (spearman coefficient: 0.73, p <0.001) and their differential activity restricted to bcr-abl-positive cells served as a positive control for the implemented test system. In vivo follow-up testing in defined tumor xenografts confirmed the results obtained ex vivo. For example, cyclophosphamide that showed strong antitumor activity with use of the NHL cell line DAUDI via clonogenic assay (IC50 = 0.3μM), also induced tumor remissions of 80% in xenografts with subcutaneously growing DAUDI cells as compared to untreated control animals. Moreover, an exceedingly promising antitumor activity of sorafenib in AML cells assessed via clonogenic assay (mean IC50 0.84μM in AML cells vs. mean IC50 4.0μM over all tested entities) could be confirmed in the disseminated in vivo model using HL-60 cells (reduction of 99% vs. untreated control; Schueler J. et al. Blood 116 (21):2141, 2010). Conclusions: The presented panel screen using clonogenic assays is of great value for time and cost effective profiling of traditional cytotoxic as well as new targeted anti-cancer agents which can be confirmed in tumor models of hematological malignancies and can thereby guide to more effectively designed in vivo experiments. Diverse activity and resistance patterns ex vivo and in vivo also contribute to create clinical development strategies of standard and novel compounds. Disclosures: No relevant conflicts of interest to declare.

The Investigational Multi-Targeted Kinase Inhibitor TAK-901 Antagonizes Acute Myeloid Leukemia Pathogenesis: Results of Preclinical Studies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 581-581
Author(s):  
Patrick Griffin ◽  
Steffan T Nawrocki ◽  
Takashi Satou ◽  
Claudia M Espitia ◽  
Kevin R. Kelly ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Standard Of Care ◽  
Clonogenic Survival ◽  
Preclinical Studies ◽  
Acute Myeloid

Abstract Abstract 581 The long-term prognosis for the majority of patients diagnosed with acute myeloid leukemia (AML) is very poor due, in part, to pre-existing myelodysplasia, multidrug resistance, and co-existing morbidities that limit therapeutic options. Novel strategies are essential in order to improve clinical outcomes. TAK-901 is an investigational small molecule kinase inhibitor that is currently being evaluated in Phase I trials. In preclinical studies, TAK-901 has demonstrated significant effects against a number of kinases with important roles in cancer including the Aurora kinases, which are key regulators of mitosis and whose overexpression in cancer promotes genetic instability, malignant pathogenesis, and drug resistance. We hypothesized that simultaneously targeting the activity of the Auroras and other oncogenic kinases with TAK-901 would disrupt AML pathogenesis. In order to test our hypothesis, we investigated the efficacy and pharmacodynamic activity of TAK-901 human AML cell lines, primary AML specimens, and an orthotopic bioluminescent disseminated mouse model of AML. TAK-901 potently diminished the viability of a panel of 8 AML cell lines as well as primary cells obtained from patients with AML. Acute exposure to TAK-901 ablated clonogenic survival, triggered the accumulation of polyploid cells, and induced apoptosis. The cytostatic and cytotoxic effects of TAK-901 were associated with significantly increased expression of the cyclin-dependent kinase inhibitor p27, growth arrest and DNA-damage-inducible 45a (GADD45a), and the BH3-only pro-apoptotic protein PUMA. Chromatin immunoprecipitation (ChIP) assays revealed that the elevation in the expression of these genes caused by administration of TAK-901 was due to increased FOXO3a transcriptional activity. The in vivo anti-leukemic activity of TAK-901 was investigated in a disseminated xenograft mouse model of AML established by intravenous injection of luciferase-expressing MV4-11 cells. IVIS Xenogen imaging was utilized to monitor disease burden throughout the study. In this mouse model, administration of TAK-901 was very well-tolerated and significantly more effective than the standard of care drug cytarabine with respect to suppressing disease progression and prolonging overall survival. Analysis of specimens collected from mice demonstrated that TAK-901 inhibited the homing of AML cells to the bone marrow microenvironment and induced AML cell apoptosis in vivo. Our collective findings indicate that TAK-901 is a novel multi-targeted kinase inhibitor that has significant preclinical activity in AML models and warrants further investigation. Disclosures: Satou: Takeda Pharmaceuticals: Employment. Hasegawa:Takeda Pharmaceuticals: Employment. Romanelli:Millennium Pharmaceuticals: Employment. de Jong:Takeda San Diego: Employment. Carew:Millennium Pharmaceuticals: Research Funding.

scholarly journals Establishment and Characterization of a Novel Dedifferentiated Chondrosarcoma Cell Line DDCS2

2021 ◽  
Vol 28 ◽  
pp. 107327482110452
Author(s):  
Xiaoyang Li ◽  
Dylan C. Dean ◽  
Al Ferreira ◽  
Scott D. Nelson ◽  
Francis J. Hornicek ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Three Dimensional ◽  
3D Culture ◽  
Growth Potential ◽  
Dedifferentiated Chondrosarcoma ◽  
Dna Profile ◽  
Chondrosarcoma Cell ◽  
Chondrosarcoma Cell Line

Background The dedifferentiated variant of chondrosarcoma is highly aggressive and carries an especially grim prognosis. While chemotherapeutics has failed to benefit patients with dedifferentiated chondrosarcoma significantly, preclinical chemosensitivity studies have been limited by a scarcity of available cell lines. There is, therefore, an urgent need to expand the pool of available cell lines. Methods We report the establishment of a novel dedifferentiated chondrosarcoma cell line DDCS2, which we isolated from the primary tumor specimen of a 60-year-old male patient. We characterized its short tandem repeat (STR) DNA profile, growth potential, antigenic markers, chemosensitivity, and oncogenic spheroid and colony-forming capacity. Results DDCS2 showed a spindle to polygonal shape and an approximate 60-hour doubling time. STR DNA profiling revealed a unique genomic identity not matching any existing cancer cell lines within the ATCC, JCRB, or DSMZ databases. There was no detectable contamination with another cell type. Western blot and immunofluorescence assays were consistent with a mesenchymal origin, and our MTT assay revealed relative resistance to conventional chemotherapeutics, which is typical of a dedifferentiated chondrosarcoma. Under ex vivo three-dimensional (3D) culture conditions, the DDCS2 cells produced spheroid patterns similar to the well-established CS-1 and SW1353 chondrosarcoma cell lines. Conclusion Our findings confirm DDCS2 is a novel model for dedifferentiated chondrosarcoma and therefore adds to the limited pool of current cell lines urgently needed to investigate the chemoresistance within this deadly cancer.

Organoids as ex vivo culture system to investigate infection-host interaction in gastric pre-carcinogenesis.

2022 ◽  
Vol 16 ◽  
Author(s):  
Cristina Di Giorgio ◽  
Rosalinda Roselli ◽  
Michele Biagioli ◽  
Silvia Marchianò ◽  
Eleonora Distrutti ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Cell Lines ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Mucosal Injury ◽  
In Vitro Models ◽  
World Population ◽  
Barr Virus

Abstract: Advancements in stem cell research have enabled the establishment of three-dimensional (3D) primary cell cultures, known as organoids. These culture systems follow the organization of an in vivo organ, as they enclose the different epithelial cell lines of which it is normally composed. Generation of these 3D cultures has bridged the gap between in vitro models, made up by two-dimensional (2D) cancer cell lines cultures, and in vivo animal models, that have major differences with human diseases. Organoids are increasingly used as a model to study colonization of gastric mucosa by infectious agents and to better understand host-microbe interactions and the molecular events that lead to infection, pathogen-epithelial cells interactions and mechanisms of gastric mucosal injury. In this review we will focus on the role of organoids as a tool to investigate molecular interactions of Helicobacter (H.) pylori and Epstein Barr Virus (EBV) and gastric mucosa and how these infections, that affect ≈ 45% of the world population, might progress to gastric cancer, a highly prevalent cancer and the third leading cause of cancer death.

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 ◽  
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.

Potent Antitumor Activity of AP24534, An Orally Active Inhibitor of Bcr-Abl, Flt3 and Other Kinases, in Both in Vitro and in Vivo Models of Acute Myeloid Leukemia (AML)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2932-2932 ◽  
Author(s):  
Victor M. Rivera ◽  
Qihong Xu ◽  
Lori Berk ◽  
Jeffrey Keats ◽  
Scott Wardwell ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Tumor Regression ◽  
Hematologic Malignancies ◽  
Fgf Receptor ◽  
Active Inhibitor ◽  
Orally Active

Abstract AP24534 is a potent, orally active inhibitor of Bcr-Abl and its mutants, including T315I, inhibiting kinase activity with IC50s of 0.3–2 nM. Potent cellular and in vivo activity of the compound has been demonstrated in models of chronic myeloid leukemia (CML). AP24534 also potently inhibits a discrete subset of other kinases, including Flt3 (IC50 13 nM), c-Kit (13 nM) and members of the FGF receptor family (2–18 nM), suggesting the potential for activity against other hematologic disorders characterized by activation of these proteins, such as acute myeloid leukemia (AML). Methods: In this study, we examined the effects of AP24534 on AML cell lines characterized by expression of various activated kinase targets, including the internal tandem duplication (ITD) variant of Flt3, FGFR1 and c-Kit. Effects on cell viability in vitro were determined using an MTS assay, and correlated with biochemical assessment of target inhibition by Western blot analysis. In vivo activity was determined by daily oral administration of AP24534 for 4 weeks in a subcutaneous tumor model using Flt3-ITD-expressing cell line MV4-11. Results: AP24534 potently inhibited the viability of AML cell lines expressing Flt3-ITD (MV4-11 cells), an activated FGFR1 fusion (KG-1 cells) or an activating c-Kit mutant N822K (Kasumi-1 cells) with IC50s of 0.7, 2.5 and 2.4 nM, respectively. In MV4-11, KG-1 and Kasumi-1 cells western blot analysis demonstrated that AP24534 inhibited the phosphorylation of the putative targets with IC50s of approximately 1, 10 and 18 nM, respectively. Furthermore, potent cellular activity (&lt;10 nM) against all 3 activated kinases was a unique characteristic of AP24534 compared with other multi-targeted kinase inhibitors tested, including sunitinib and sorafenib. In vivo activity of AP24534 was examined in an MV4-11 mouse xenograft model. Statistically significant inhibition of tumor growth was demonstrated with once-daily oral doses as low as 1 mg/kg, and partial tumor regression with doses of 2.5 mg/kg. Doses of 5 or 10 mg/kg led to complete and durable tumor regression with no palpable tumors detected during a 4 week follow-up period. A single 10 mg/kg dose of AP24534 was sufficient to block phosphorylation of STAT5, a major downstream target of Flt-3. These potencies and responses observed in AML cell lines are comparable to the observed effects of AP24534 in analogous in vitro and xenograft studies using the Bcr-Abl-driven CML cell line K562. Conclusions: These results indicate that AP24534 has the potential to be an effective treatment for AML, including the approximately one-third of AML cases characterized by the Flt3- ITD mutation that is correlated with a poor prognosis. The compound was particularly potent on the Flt3-driven cell line MV4-11. Inhibition by AP24534 of non-Flt3-dependent AML cell lines, such as those driven by c-Kit or FGF receptors, indicates the potential for activity across diverse AML subtypes and other c-Kit or FGF receptor-driven malignancies, such as multiple myeloma. Together with previous data showing potent activity in CML models, these results suggest a broad potential for AP24534 in hematologic malignancies. Based on these observations, a phase 1 clinical trial is now underway to evaluate AP24534 in patients with a range of hematologic malignancies, including AML and CML.

Preclinical Efficacy and Toxicology Studies of APR-246, a Novel Anticancer Compound Currently In Clinical Trials for Refractory Hematological Malignancies and Prostate Cancer

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1806-1806
Author(s):  
Nina Mohell ◽  
Charlotta Liljebris ◽  
Jessica Alfredsson ◽  
Ylva Lindman ◽  
Maria Uustalu ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Ex Vivo ◽  
Primary Cells ◽  
In Vivo Efficacy ◽  
Preclinical Safety ◽  
Anticancer Compound

Abstract Abstract 1806 The tumor suppressor protein p53 is a transcription factor involved in cell cycle arrest, senescence and apoptosis. The p53 gene is frequently mutated in cancer, and cancer cells carrying defects in p53 are generally more resistant to conventional chemotherapy. Thus, restoration of wild type function of p53 is a promising novel strategy for cancer therapy. APR-246 belongs to a new class of small molecules (quinuclidinones) that reactivates non-functional p53 by promoting its correct folding and triggering apoptosis (Lambert et al. Cancer Cell 15, 2009). The lead compound of APR-246, PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis) was identified by a cellular screen of a NCI (National Cancer Institute) library, and an optimization program led to the discovery of the analog APR-246 (PRIMA-1MET). In various in vitro,ex vivo andin vivo cancer models, APR-246 has shown good antitumor activity. It reduces cell viability and/or induces apoptosis in a large number of human cancer cell lines with different p53 status, including leukemia, lymphoma and myeloma cell lines (Mohell et al. Blood 114, 2009). Ex vivo efficacy of APR-246 alone and in combination with conventional chemotherapeutic drugs has been shown in primary cells from patients with acute myeloid leukemia (AML) (Jonsson-Videsater et al. Blood 114, 2009). Ex vivo efficacy of APR-246 has also been shown in primary cells from patients with chronic lymphocytic leukemia (CLL). APR-246 was 4–8 fold more potent in killing malignant than normal lymphocytes, whereas common cytostatics often have negative ratio (Mohell et al. Blood 114, 2009). In vivo efficacy of APR-246/PRIMA-1 has been demonstrated in xenograft studies using many solid tumor cell lines (Mohell et al. Blood 114, 2009). Here we present results from studies with APR-246 using in vivo systemic and metastasic xenograft model with the human AML primary cell line AML-PS. This model was established by Giovazzi et al. (Int. J. Cancer 61, 1995) and is considered as a predictive in vivo model for human AML. In addition, some key results from preclinical safety and toxicology studies are reported. Briefly, SCID (severe combined immunodeficiency) mice were inoculated i.v. with 5×106 human AML-PS primary cells. Three days after inoculation treatment with i.v. injections of APR-246 (200 and 300 mg/kg), twice daily for 10 days, was initiated. Mice were monitored daily for health status and mortality. Blood samples were collected for determination of the percentage of circulating human leukemia cells by FACS analysis. Human leukemic cells were detected using a fluorescent antibody against the major histocompatibility complex (HLA). In parallel, pharmacokinetic experiments to measure the concentration of APR-246 in the blood were performed. We found that APR-246 had a statistically significant antitumor effect by decreasing the percentage of circulating human AML-PS cells and increasing the survival time of the mice (P=0.0024, n=10). A good correlation between increase in survival time and decrease in circulating tumor cells in the blood was observed. Further in vivo efficacy studies using various treatment schedules and combinations with conventional cytostatics are ongoing. APR-246 was also investigated in pivotal toxicology studies using single and repeat-dose regimen. In dogs, APR-246 was well tolerated when administered as 2 h infusion with NOAEL (no observed adverse effect level) of 200 mg/kg/day (4000 mg/m2/day). In both dogs and mice, Cmax levels less than 100 μg/ml did not induce any toxicity, regardless of the administration protocol. No systemic target organ toxicity was observed in mice or dogs, including blood and bone-marrow parameters. In conclusion, APR-246 has in various efficacy models demonstrated significant antitumor activity and a unique pharmacological profile. In preclinical safety/toxicity studies no toxicity at predicted therapeutic plasma concentrations was observed. Thus, APR-246 appears to be a promising novel anticancer compound to treat patients resistant to common chemotherapy. Currently, APR-246 is investigated in a dose escalating Phase I/IIa First-in-Man study for refractory hematological malignancies and prostate cancer. The Phase II Proof of Concept study is planned to start in 2011. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Uhlin:Aprea AB: Employment. Linderholm:Aprea AB: Consultancy. Wiman:Aprea AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Profound Biological Difference of Chronic and Blast Phase Chronic Myeloid Leukemia in Ex Vivo Drug Responses

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3139-3139
Author(s):  
Paavo Pietarinen ◽  
Tea Pemovska ◽  
Emma I Andersson ◽  
Perttu Koskenvesa ◽  
Mika Kontro ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Primary Cells ◽  
Newly Diagnosed ◽  
Biological Difference ◽  
Resistant Patient

Abstract BACKGROUND Most patients with chronic phase (CP) chronic myeloid leukemia (CML) are successfully treated with tyrosine kinase inhibitors (TKIs) targeting ABL1. Despite the good results, TKI treatment rarely results in cure, and some patients relapse and progress to advanced phases of CML. Accelerated phase and blast crisis (BC) have remained a therapy challenge. We set out to identify novel candidate drugs for chronic and advanced phase CML by using an unbiased high-throughput drug testing platform and utilizing both primary patient cells (CP and BC) and cell lines. METHODS CML BC cell lines used: K562 (erythroleukemic), MOLM-1 (megakaryocytic) and EM-2 (myeloid). Primary bone marrow (BM) and peripheral blood (PB) samples were derived from 3 CML patients with BC, two of which were TKI-resistant. Patient 1 had developed resistance to imatinib and nilotinib due to an E274K mutation in ABL1 kinase domain, whereas patient 2 was resistant to imatinib, nilotinib, and dasatinib due to a T315I mutation. In addition to BC patients, samples from 23 newly diagnosed CML CP patients were screened. BM cells from 4 healthy individuals were used as controls. Functional profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 306 anti-cancer agents. Cells were dispensed to pre-drugged 384-well plates and incubated for 72 h. Cell viability was measured using a luminescence cell viability assay (CellTiter-Glo, Promega). A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. The drug sensitivities of the primary cells were further normalized against the median values from healthy controls, resulting in leukemia-specific sensitivity scores (sDSS). RESULTS Drug sensitivities of CML cell lines correlated closely (EM-2 vs. K-562, rS=0.89; EM-2 vs. MOLM-1, rS=0.82; K-562 vs. MOLM-1, rS=0.78; p<0.0001 for all correlations). Similarly, patient samples had good correlation with cell line samples (rS=0.82 based on median values; p<0.0001). The cell lines were highly sensitive to ABL1-targeted TKIs, with the exception of the MOLM-1, which showed only modest sensitivity (Figure). The clinically TKI-resistant patient samples were also resistant to BCR-ABL1 inhibitors ex vivo (e.g. T315I sensitive only to ponatinib), further validating the DSRT assay data. Other drugs that exhibited high DSS in the CML cell lines and high sDSS in the BC patient samples included mTORC1/2 inhibitors (e.g. AZD8055, AZD2014, INK128), HSP90 inhibitors (e.g. NVP-AUY922, BIIB021) and a NAMPT inhibitor daporinad. Remarkably, the DSRT results from newly diagnosed CML CP differed clearly from those derived from the cell line and CML BC samples. In the clustering analysis, CML BC and cell line samples clustered together, whereas CML CP samples formed a separate group (Figure). The leukemia-specific scores were generally much lower in CML CP samples, which made identifying novel candidate compounds challenging. Most surprisingly the responses to TKIs were practically nonexistent in CML CP samples. CP TKI insensitivity was further assessed with primary cells sorted in CD34pos and CD34neg fractions. Preliminary results from two patients suggested that CD34pos cells were more sensitive to TKIs when compared to CD34neg or whole mononuclear fraction. CONCLUSIONS DSRT is a powerful platform for identifying novel candidate molecules for CML BC patients. Our results indicate that mTORC1/2 inhibitors (such as AZD8055, or AZD2014), HSP90 (such as NVP-AUY922/luminespib) and NAMPT inhibitors in particular warrant further clinical evaluation. TKI-insensitivity of CML CP samples suggests that the survival of mature myeloid cells in vitro is not BCR-ABL1 dependent and reflects a clear biological difference between CP and BC patient cells. Figure 1 Figure 1. Disclosures Kallioniemi: Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3107-3116 ◽  
Author(s):  
J Tsuchiyama ◽  
M Mori ◽  
S Okada
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Mouse Spleen ◽  
Hematopoietic Organ

The hematopoietic microenvironment (HIM) of mouse spleen predominantly induces the differentiation of hematopoietic progenitors into erythroid lineage in vivo. However, the mechanisms of this phenomenon have not been fully explored because of the lack of an adequate in vitro system mimicking the spleen hematopoiesis. To reconstruct the HIM of mouse spleen in vitro, we established spleen stromal cell lines from a three-dimensional (3D) spleen primary culture in collagen gel matrix. Of these, SPY3–2 cells were negative for preadipocytic and endothelial markers, had a fibroblastoid morphology, and were not converted to adipocytes in the presence of 1 mumol/L hydrocortisone. They supported the maintenance and multilineal differentiation of hematopoietic progenitor cells for more than 8 weeks in vitro. The differentiated hematopoietic cells in the coculture medium were predominantly monocytes rather than granulocytes. Furthermore, erythropoiesis was predominantly induced in the presence of 2 U/mL erythropoietin and continued for more than 12 weeks. The number of burst-forming units-erythroid (BFU-E) was increased 10 times after 3 weeks of coculture, which was followed by pronounced production of erythroid cells in the coculture after week 4. SPY3–2 expressed high levels of c-kit ligand and low levels of granulocyte macrophage colony-stimulating factor and interleukin-3, and these molecules were all involved in this long-term erythropoiesis. Thus, the clonal SPY3–2 cell line will provide a novel HIM in vitro analogous to that of mouse spleen in vivo. These results suggest that 3D collagen gel culture may facilitate the establishment of functioning stromal cell lines of hematopoietic organ.

Pharmacological Validation Of Potentiating Targets From SAHA RNA-Interference Modifier Screens In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3832-3832
Author(s):  
James M Bogenberger ◽  
James Rudd ◽  
Donald Chow ◽  
Michelle Kassner ◽  
Veena Fauble ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Rna Interference ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Hdac Inhibition ◽  
Myeloid Malignancies ◽  
Rational Combination ◽  
Acute Myeloid

Abstract The lysine deacetylase inhibitor suberoylanilide acid (SAHA) exhibits promising but limited activity in the treatment of acute myeloid leukemia (AML). To identify potential targets for rational combination therapies that increase the efficacy of SAHA in AML, we performed a functional RNA-interference (RNAi) drug modifier screen to identify genes that, when inhibited, potentiate or antagonize the in vitro anti-leukemic activity of SAHA. A total of 901 kinase, phosphatase and closely associated signaling genes were silenced in TF-1, HEL and THP-1 cells, with four different siRNA sequences per gene, both alone and in combination with SAHA treatment. Screen hit lists for each cell line were over-laid on an integrated functional relationship network. A community detection algorithm was then applied to this sub-network and siRNA sensitive modules were identified. Each module represents a highly connected set of genes in the integrated network. To identify pathways represented by each module, enrichment was evaluated using the National Cancer Institute (NCI) Protein Interaction Database (PID) pathways. Both cell line-specific and universal sensitizing targets, grouped into a small number of pathways, emerged from these screens. Seven pharmacologic inhibitors interfering with these pathways have been assessed in a panel of four or five AML cell lines, including SET-2, TF-1, HEL, THP-1, and OCI-AML3. Of the seven tested inhibitors targeting SAHA sensitizing networks, one combination exhibiting universal SAHA sensitization in all AML cell lines tested was further assessed in drug dose response assays using ex vivo cultures of bone marrow mononuclear cell populations derived from patients with various myeloid malignancies. The combination demonstrated “one-sided” SAHA EC50 fold-enhancement, as well as strong synergy by “two-sided” CalcuSyn combination index determination in all ex vivo specimens examined to date (N=6), including polycythemia vera (PV), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndrome, MDS transformed to AML, and de novo AML samples. Thus, this first reported large-scale SAHA RNAi modifier screen in AML has identified a specific pathway/target whose inhibition broadly synergizes with HDAC inhibition in myeloid malignancies. A novel compound putatively targeting this respective pathway/target is currently in early clinical development and has shown limited single-agent activity. With the full dataset presented, we will propose a novel rational combination to enhance the activity of HDAC inhibition in AML and other myeloid malignancies that could be translated into design of a clinical trial. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Alkylating Histone Deacetylase Inhibitor Fusion Molecule Edo-S101 Displays Full Bi-Functional Properties in Preclinical Models of Hematological Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2100-2100 ◽  
Author(s):  
Ana Alicia López-Iglesias ◽  
Laura San-Segundo ◽  
Lorena González-Méndez ◽  
Susana Hernández-García ◽  
Daniel Primo ◽  
...  
Keyword(s):  
Single Dose ◽  
Cell Line ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Parp Cleavage ◽  
Hdac Inhibition ◽  
Histone 3 ◽  
Xenograft Models

Abstract Background Alkylating histone deacetylase inhibitors (HDACi) enhance the anticancer efficacy of alkylators by increasing chromatin accessibility and also down regulating DNA repair. EDO-S101 is a first-in-class fusion molecule that combines DNA damaging effect of bendamustine with the pan-HDACi vorinostat. Objectives To study the bi-functional properties of EDO-S101 as an alkylating agent and a pan-HDACi in various in vitro and in vivo xenograft models of hematological malignancies. Methods In vitro inhibition of HDAC Class I and II enzymes by EDO-S101 and vorinostat was tested using an recombinant human enzymatic assay (BPS Bioscience, Enzo Life Science) and in vivo in rat peripheral blood mononuclear cells (PBMCs). The degree of inhibition was measured 1 hour following a single dose of 10–50 mg/kg i.v. and duration of inhibition over 24 hours after a single i.v. dose of EDO-S101 of 25 mg/kg. HDAC inhibition, alkylation and apoptotic activity were evaluated in vitro in myeloid (HL60 AML cell line) and lymphoid cell lines, including Daudi Burkitt’s lymphoma (BL) and a panel of 6 MM cell lines (MM1S, MM1R, RPMI-8226, RPMI-LR5, U266, U266-LR7). In vivo intra-tumor effects were analyzed after short courses of treatment with EDO-S101 in MM1S human plasmacytoma (PC) and BL xenograft models. Changes in pathway activation, protein expression and activities influencing the cell cycle were measured by Western blot and immunohistochemistry. Anti-tumor activity in vitro was measured by MTT and in vivo using a caliper to assess tumor size at regular intervals. Results In vitro, EDO-S101’s pan-HDACi activity, at nanomolar concentrations in Class I and II recombinant enzymes, was similar to vorinostat. In vivo, in intact rat PBMCs, HDAC inhibition was maximal at 1 hour after a single dose of 10 mg/kg i.v.–the dose where antitumor activity starts. HDAC inhibition did not increase with doses up to 50 mg/kg, recovery began within 3 hours and was nearly complete at 16 hours. In the AML HL60 cell line in vitro, hyperacetylation of lysine residues K9, K14, K23 and K56 on histone 3 was found after exposure to 2–4 µM of EDO-S101. Histone 3 and 4 hyperacetylation was also demonstrated in MM cell lines at 1–5 µM concentrations. In xenograft models of human plasmacytoma and BL, EDO-S101 induced histone 3 hyperacetylation, indicating an HDACi effect in vivo. Alkylating activity was demonstrated in vitro in HL60 and MM cell lines by DNA cross-linking and double strand break formation in the comet assay by immunofluorescence. In vivo, in xenograft models of human plasmacytoma (60 mg/kg d 1, 8, 15) and BL (40 and 80mg/kg d1) exposure to EDO-S101 caused a strong DNA-repair response shown by activation of pH2AX and p53 (PC and BL) followed by an increase of DNA damage check point proteins pCHK1 (PC) and even more prominent pCHK2 (PC and BL). The kinetics of this effect, studied in vivo in BL tumors, showed that the pH2AX response fell at Day 8 after dosing while the p53 response lasted, particularly in the group treated with 80mg/kg. In Daudi-bearing mice tumors, p-ATR was completely suppressed at Day 8 after treatment, which was not clear in the PC tumors. EDO-S101 triggered apoptosis in vitro and in vivo, resulting in strong antitumor activity in HL60, Daudi and the panel of six MM cell lines. Initial in vitro experiments in HL60 cells showed an activation of the intrinsic pathway of apoptosis with cleavage of caspases 3, 9 and PARP and a marked reduction of anti-apoptotic proteins XIAP and Mcl-1. In the MM cell line, MM1S activation of the intrinsic and extrinsic pathways of apoptosis (C 8, 9, 3, 7 and PARP cleavage) was seen with a loss of mitochondrial membrane potential by DiOC6. Tumors of human plasmacytoma and BL in vivo were rapidly shrinking or completely eradicated after i.v. administration of EDO-S101. A decrease in proliferation (Ki67) and slight PARP cleavage was found in the tumor tissue (PC), and evidence of activation of apoptosis by cleavage of caspases 7 and 9 at Day 4 and caspase 8 and PARP at Day 8 after treatment in BL tumors. The level of caspase 3, different to MM, remained unchanged. Importantly, EDO-S101 induced a rapid and dose-dependent strong decrease of XIAP and Mcl-1 which lasted until Day 8. Conclusions This study demonstrates the bi-functional mechanism of ED0-S101 in both myeloid and lymphoid hematological malignancies. The data support the clinical investigation of EDO-S101 in treating hematological malignancies. Disclosures Ocio: Mundipharma: Honoraria, Research Funding. Mehrling:Mundipharma: Employment.

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