scholarly journals Derived from a Platform of Innovative Therapeutic Molecular Clusters (TMC), Ag5 Is a Highly Potent and Differentiated Molecule That Promises to be Efficacious in Hard-to-Treat Hematological Cancers

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 23-23
Author(s):  
Vanesa Porto González ◽  
Carmen Carneiro ◽  
Vanesa Santos ◽  
Erea Borrajo ◽  
Blanca Dominguez ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Residual Disease ◽  
Water Soluble ◽  
Molecular Cluster ◽  
Medical Need ◽  
Wide Range ◽  
Hematological Cancers

[Background on medical need in hematology]Even though significant progress has been made in a number of hematological malignancies in recent years, an unmet medical need still remains in indications such as multiple myeloma (MM), chronic lymphocytic leukemia (CLL) or acute myeloid leukemia (AML) due to refractory disease, severe adverse events or the failure to achieve sustained minimal residual disease. As a consequence, novel therapies are required to prevent relapse, be safer in administration and to target residual disease. We present here for the first time a novel and innovative therapeutic approach with the potential to treat patients suffering from hard-to-treat hematological malignancies based on the tumor´s pathophysiology and reactive oxygen species (ROS) phenotype. [Background on Ag5]TMC are created by combining the atoms of certain transition metals under specific conditions to form novel molecules with entirely distinct properties from "traditional" metal. For example, silver can be used to create clusters of different and defined sizes, based on the number of atoms making up the final drug candidate. More specifically, Ag5 contains five silver atoms arranged in a specific conformation, is the first TMC derived from this novel platform and has entirely different physiological properties than the three silver atom containing Ag3. Ag5 is a water-soluble and heat stable molecule, it is orally bioavailable and a freely diffusible pan-tumor therapeutic. It selectively kills those cells with high ROS concentrations by oxidizing their antioxidant systems and subsequently drives these cells to programmed cell death. In consequence, Ag5 will preferentially kill cancer cells which typically have higher ROS levels, but will spare normal cells which display lower ROS due to their functional REDOX homeostasis. More specifically, Ag5 efficiently catalyzes the oxidation of thiol groups of thioredoxins and peroxiredoxins and thereby drives sensitive cells above a threshold to irreversible protein misfolding, protein degradation and programmed cell death. [Background on Ag5 experiments]We characterized Ag5 efficacy using a wide range ofin vitroassays and found potent Ag5 efficacy against a number of MM, CLL and AML cell lines with an IC50 in the low nM range. Ag5 sensitivity of all cell lines was correlated with ROS levels, more specifically superoxide, as measured by dihydroethidium (DHE) or MitoSOX. Furthermore, we were able to demonstrate that Ag5 treatment resulted in a concentration dependent cell cycle arrest in G1 phase, mitochondrial swelling and induction of apoptosis. Treatment of primary CLL tumor samples resulted in low nM efficacy. Finally, we could demonstrate that Ag5 was not only safely administered without any side effects in mouse and rat studies, but was equally effective as the stand-of-care bortezomib in a multiple myelomain vivomodel. [Conclusion and clinical significance]In summary, Ag5 is a novel and innovative therapeutic candidate that was shown to be safe and effective in preclinical studies, and has the promise to address the unmet medical need in hard-to-treat hematological malignancies. Keywords: Ag5, Therapeutic molecular cluster (TMC), Redox, ROS, catalysis, Ag3, AML, MM, CLL Disclosures Porto González: Arjuna:Research Funding.Carneiro:Arjuna:Research Funding.Lopez-Quintela:Arjuna:Current equity holder in private company.Treder:Arjuna:Current Employment.Dominguez:Arjuna:Current equity holder in private company.

scholarly journals Comparative evaluation of the effectiveness of methods for certification of transplanted cell lines

2021 ◽  
Author(s):  
E.V. Markova ◽  
V.T. Nochevny ◽  
B.L. Manin ◽  
I.N. Matveeva
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Cell Lines ◽  
Traditional Method ◽  
Biological Properties ◽  
International Standards ◽  
Growth Potential ◽  
Wide Range

The article presents the results of certification of two trofovariants of MDVK cell lines with the help of traditional method and flow cytometry. Research object was the test cultures MDBK-E and MDBK-B, which passed 30 and 43 passages, respectively, after cryopreservation. The traditional method of attestation of transplanted cell lines, widely used in practice, is rather laborious and requires significant expenditures of labor, money and time. The flow cytometry method is based on a wide range of cytochemical and fluorescent methods for the analysis of sizes, granularity, phases of the cell cycle, structural components (DNA, RNA, protein), cell apoptosis and a number of other indicators. It was experimentally established that the sublines of MDBK-E and MDBK-B cells differed in cultural, cytomorphological and karyological parameters, as well as in contamination by foreign agents and sensitivity to parainfluenza-3 viruses and infectious rhinotracheitis in cattle. Analysis of histograms of cell distribution depending on the DNA content showed that the studied lines MDBK-E and MDBK-B did not exceed the standard indicator in terms of apoptosis and were at the level of 3,9 and 6,8%, respectively. Cells of the MDBK-E line did not contain viral and mycoplasma contamination, were characterized by a pronounced growth potential, retained the original cell morphology and were the most promising substrate for the production of antigens of parainfluenza-3, infectious rhinotracheitis in cattle. Analysis of granularity distribution results testified to the violation of the division processes and the appearance in the population of the subline MDBK-B of abnormal cells, as well as inadequate conditions for maintaining the test culture. It has been established that the flow cytometry method is objective and quite promising in the selection of culture models that meet the requirements of domestic and international standards. The revealed correlation between the magnitude of apoptosis, cultural properties and parameters of the cell cycle makes it possible to assess the biological properties of the producer culture as one of the leading factors in the change in programmed cell death. Changes the index of programmed cell death underlies a number of important pathological conditions and degenerative processes.

Stapled Peptides: Leveraging the BH3 α-Helix to Create a New Class of Drugs to Treat Hematological Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2929-2929
Author(s):  
Rosana Kapeller ◽  
Jiawen Han ◽  
Kaiming Sun ◽  
Pranoti Gangurde ◽  
Nori Kawahata ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Cancer Cells ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Lung Fibroblasts ◽  
Resting Cells ◽  
Stapled Peptides ◽  
Selective Approach

Abstract AILERON is developing a selective approach to restore programmed cell death in cancer cells by borrowing from nature the BH3 domain alpha-helical “keys” from pro-apoptotic members of the BCL-2 family that fit into the “locks” of other anti- and pro-apoptotic BCL-2 proteins. AILERON has applied its proprietary chemical strategy termed hydrocarbon stapling (J. Am. Chem. Soc., 2000 122:5891) to generate cell-permeable BH3 stapled peptides that modulate the intracellular protein-protein interactions of BCL-2 family members and selectively kill cancer cells both in vitro and in vivo (Science, 2004 305:1466). The lead compounds possess the innate characteristics of the endogenous peptides they mimic, including mechanism of action and target specificity. BID BH3 and BIM BH3 stapled peptides have recently been shown to directly activate the pro-death molecule BAX, a unique property that differentiates BH3 stapled peptides from all BCL-2 small molecule antagonists (Mol. Cell, 2006 24:199). BID BH3 and BIM BH3 stapled peptides were tested for their ability to induce programmed cell death in a panel of 12 lymphoid-derived tumor cell lines. T-ALL derived cell lines were the most sensitive to the compounds, followed by multiple myeloma lines. CML-derived cell lines were the least sensitive. Resting human peripheral blood lymphocytes (hPBLs) and normal human embryonic lung fibroblasts (WI-38) were found to be resistant to BH3 stapled peptides, indicating that the compound may target only cells that are “primed to die” and not normal “resting” cells. The anti-cancer activity of BH3 stapled peptides was further investigated in orthotopic xenograft models and shown to dramatically suppress tumor growth. In a mixed lineage leukemia model (SEMK2), a tumor over control (T/C) of 27% was observed after 13 days of treatment at 30 mg/Kg IV q.d. We also investigated whether BH3 stapled peptides elicit an antibody response in rodents. No antibody titer was detected, indicating that BH3 stapled peptides are non-antigenic in rodents. While peptides are oftentimes unstable in vivo, with half-lives typically in the range of a few minutes, BH3 stapled peptides were 100% stable in both mouse and human plasma ex vivo, and exhibited excellent PK profiles in rats with half-lives greater than three hours. BH3 stapled peptides were well tolerated in all animal models tested to date. In conclusion, we show that BH3 stapled peptides exhibit promising pharmacological properties and represent a novel class of drugs for the treatment of hematological malignancies.

CAL-101, a Potent Selective Inhibitor of the p110d Isoform of Phosphatidylinositol 3-Kinase, Attenuates PI3K Signaling and Inhibits Proliferation and Survival of Acute Lympoblastic Leukemia in Addition to a Range of Other Hematological Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 16-16 ◽  
Author(s):  
Brian J Lannutti ◽  
Sarah A. Meadows ◽  
Adam Kashishian ◽  
Bart Steiner ◽  
Sarah May ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
B Cell Lymphoma ◽  
Class I ◽  
Pi3k Signaling ◽  
Wide Range ◽  
Normal Human ◽  
Phosphatidylinositol 3

Abstract The class I phosphatidylinositol 3-kinases (PI3K) regulate a variety of cellular functions including motility, metabolism, proliferation, growth, and survival, depending on cellular milieu. Deregulation of the PI3K/Akt pathway is one of the most frequently observed defects in human malignancies including those of hematological origin and has been shown to play an important role in tumor progression. Therefore, selective targeting of PI3K signaling in hematological tumor cells could provide an effective treatment strategy while limiting potential undesirable effects of pan-inhibitors that broadly block PI3K signaling in all cells. Of the class IA PI3Ks (p110a, p110b, p110d), p110d’s expression is largely restricted to cells of hematopoietic origin and is essential for PI3K signaling in lymphocytes. Here, we report on the characterization of a novel p110d specific inhibitor, CAL-101. This compound is a potent PI3K inhibitor with an IC50 of 1–10 nM against the purified p110d subunit and 30–70 nM cellular potency against p110d-mediated basophil activation in whole blood. Importantly, CAL-101 plasma concentrations of 500–5000 nM that greatly exceed those needed for p110d inhibition in blood were safely maintained in a 7 day multidose normal human volunteer study. CAL-101 demonstrates >30-fold selectivity over other class I, II and III PI3K family members as well as selectivity over other PI3K-related proteins including mTOR and DNA-PK. Furthermore, a genome wide screen of >350 protein kinases did not detect any activity. To investigate the potential role of p110d in hematologic tumors we screened a wide range of leukemia and lymphoma cell lines for p110 isoform expression and constitutive PI3K pathway activation. The expression of p110d was observed in >90% of these cell lines that was in many cases accompanied by constitutive Akt phosphorylation. In this context, CAL-101 was able to reduce p-Akt levels and block additional downstream effectors such as p-p70S6K, p-GSKb, and p-Bad in cells that represent a range of tumor types including acute myeloid leukemia, acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma among others. Recent studies have demonstrated the importance of PTEN loss and enhanced PI3K signaling in primary T-ALL cells. We report high levels of p110d protein and activated Akt in 6 of 6 ALL cell lines evaluated. Inhibition of p110d with CAL-101 treatment of both T-ALL and B-ALL cell lines resulted in a reduction of Akt and GSK-3b phosphorylation and a decrease in cellular proliferation that was accompanied by cell death demonstrating an essential role of PI3K signaling independent of PTEN status. Treatment of T-ALL cell lines with CAL-101 induced processing of pro-caspase-3 and cleavage of PARP supporting a role for caspase mediated cell death. These studies have now been extended to the analysis of primary patient blast samples to further establish preclinical proof of concept for therapeutic application of CAL-101 for the treatment of ALL. In summary, CAL-101 is a highly potent and selective p110d kinase inhibiter with broad anti-tumor activity against cancer cells of hematologic origin. Clinical studies in normal human volunteers demonstrated good tolerability with high drug exposure and favorable steady-state pharmacokinetic properties. Taken together, these data support the on going Phase 1 clinical trial that includes a wide range of hematological malignancies.

scholarly journals Induction of programmed cell death in human hematopoietic cell lines by fibronectin via its interaction with very late antigen 5.

1994 ◽  
Vol 179 (6) ◽  
pp. 1757-1766 ◽  
Author(s):  
H Sugahara ◽  
Y Kanakura ◽  
T Furitsu ◽  
K Ishihara ◽  
K Oritani ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Cell Lines ◽  
Growth Suppression ◽  
Hematopoietic Cell ◽  
Leukemia Cell Line ◽  
Gm Csf ◽  
Late Antigen ◽  
Antigen 5 ◽  
Hematopoietic Cell Lines

Extracellular matrix (ECM) molecules such as fibronectin (FN), collagens, and laminin have important roles in hematopoiesis. However, little is known about the precise mechanisms by which ECM molecules regulate proliferation of human hematopoietic progenitor cells. In this study, we have investigated the effects of ECM molecules, particularly of FN, on the proliferation of a myeloid leukemia cell line, M07E, which proliferates in response to either human granulocyte/macrophage colony-stimulating factor (GM-CSF) or stem cell factor (SCF). The [3H]thymidine incorporation and cell enumeration assays showed that FN strikingly inhibited GM-CSF- or SCF-induced proliferation of M07E cells in a dose-dependent manner, whereas little or no inhibition was induced by collagen types I and IV. The growth suppression of M07E cells was not due to the inhibitory effect of FN on ligand binding or very early events in the signal transduction pathways from the GM-CSF or SCF receptors. DNA content analysis using flow cytometry after staining with propidium iodide revealed that the treatment of M07E cells with FN did not block the entry of the cells into the cell cycle after stimulation with GM-CSF or SCF, whereas the treatment resulted in the appearance of subdiploid peak. Furthermore, FN was found to induce oligonucleosomal DNA fragmentation and chromatin condensation in the cells even in the presence of GM-CSF or SCF, suggesting the involvement of programmed cell death (apoptosis) in the FN-induced growth suppression. The growth suppression or apoptosis induced by FN was rescued by the addition of either anti-FN antibody, anti-very late antigen 5 monoclonal antibody (anti-VLA5 mAb), or GRGDSP peptide, but not by that of anti-VLA4 mAb or GRGESP peptide, suggesting that the FN effects on M07E cells were mediated through VLA5. In addition, the FN-induced apoptosis was detectable in VLA5-positive human hematopoietic cell lines other than M07E cells, but not in any of the VLA5-negative cell lines. These results suggest that FN is capable of inducing apoptosis via its interaction with VLA5, and also raise the possibility that the FN-VLA5 interaction may contribute, at least in part, to negative regulation of hematopoiesis.

Mitochondrial DNA–Related Mitochondrial Dysfunction in Neurodegenerative Diseases

2002 ◽  
Vol 126 (3) ◽  
pp. 271-280
Author(s):  
Russell H. Swerdlow
Keyword(s):  
Cell Death ◽  
Mitochondrial Dna ◽  
Programmed Cell Death ◽  
Neurodegenerative Diseases ◽  
Mitochondrial Dysfunction ◽  
Cell Lines ◽  
Late Onset ◽  
Cell Death Pathways ◽  
Mitochondrial Genotype ◽  
Cybrid Cell

Abstract Mitochondrial dysfunction occurs in several late-onset neurodegenerative diseases. Determining its origin and significance may provide insight into the pathogeneses of these disorders. Regarding origin, one hypothesis proposes mitochondrial dysfunction is driven by mitochondrial DNA (mtDNA) aberration. This hypothesis is primarily supported by data from studies of cytoplasmic hybrid (cybrid) cell lines, which facilitate the study of mitochondrial genotype-phenotype relationships. In cybrid cell lines in which mtDNA from persons with certain neurodegenerative diseases is assessed, mitochondrial physiology is altered in ways that are potentially relevant to programmed cell death pathways. Connecting mtDNA-related mitochondrial dysfunction with programmed cell death underscores the crucial if not central role for these organelles in neurodegenerative pathophysiology. This review discusses the cybrid technique and summarizes cybrid data implicating mtDNA-related mitochondrial dysfunction in certain neurodegenerative diseases.

Aldehyde biphenyl chalcones induce immunogenic apoptotic-like cell death and are promising new safe compounds against a wide range of hematologic cancers

2020 ◽  
Vol 12 (8) ◽  
pp. 673-688
Author(s):  
Mariana F Maioral ◽  
Natália M Stefanes ◽  
Patrícia D Neuenfeldt ◽  
Louise D Chiaradia-Delatorre ◽  
Ricardo J Nunes ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Endoplasmic Reticulum Stress ◽  
Hematological Malignancies ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Promyelocytic Leukemia Protein ◽  
Wide Range ◽  
Antileukemic Effect

Aim: Investigate the apoptotic mechanisms of two new aldehyde biphenyl chalcones on leukemia cells. Materials & methods: From a series of 71 new chalcones, we selected the two most cytotoxic. Results: JA3 and JA7 were cytotoxic not only against hematological malignancies but also against solid tumor and cancer stem cells, yet with no toxicity to normal cells. Moreover, they induced immunogenic apoptotic-like cell death independently of promyelocytic leukemia protein, with extensive mitochondrial damages downstream of endoplasmic reticulum stress. Preventing endoplasmic reticulum stress and the upregulation of proapoptotic machinery inhibited JA3- and JA7-induced cell death. Likewise, blocking receptor Fas protected cells from killing. They increased the antileukemic effect of cytarabine and vincristine and killed leukemic cells collected from patients with different acute leukemia subtypes. Conclusion: JA3 and JA7 represent new promising prototypes for the development of new chemotherapeutics.

Degradation of Aiolos and Ikaros Followed By Downregulation of c-Myc and then IRF4 Leads to Programmed Cell Death in Myeloma Cells Sensitive to IMiDs® Immunomodulatory Compounds

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4700-4700
Author(s):  
Chad C Bjorklund ◽  
Courtney G. Havens ◽  
Patrick R Hagner ◽  
Anita K. Gandhi ◽  
Maria Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Protein Expression ◽  
Cell Lines ◽  
Acquired Resistance ◽  
Clinical Samples ◽  
Employment Equity ◽  
Molecular Sequence ◽  
Sequence Of Events ◽  
Equity Ownership

Abstract Background: The zinc finger transcription factors, Aiolos (IKZF3) and Ikaros (IKZF1) were identified as lenalidomide (LEN) and pomalidomide (POM)-induced substrates of the cereblon (CRBN)-dependent Culin4 E3-ligase complex. While recent studies suggest that the anti-proliferative activity of LEN and POM in multiple myeloma (MM) cell lines in vitro is due in part to the targeted ubiquitination and subsequent proteasomal degradation of Aiolos and Ikaros, the downstream molecular mechanisms remain unknown. Using inducible shRNA-mediated knockdown combined with kinetic analyses, we systematically investigated the biological mechanisms associated with the degradation of Ikaros and Aiolos in MM cell lines that are sensitive to or have acquired resistance to LEN and POM. Results: In MM1.S and U266 MM cell lines stably engineered with doxycycline (DOXY)-inducible shRNAs, knockdown of either Ikaros or Aiolos showed a reduction in cell proliferation (80%-90%) as measured by 3H-thymidine incorporation after a 4 day treatment with DOXY. We demonstrated that this anti-proliferative effect is inherently tied to and precedes the induction of apoptosis, which was maximized (60%-80% AnnV+/ToPro3+) 5 days following Aiolos or Ikaros knockdown compared with a control shRNA. shRNA-mediated knockdown of Aiolos or Ikaros was furthermore associated with decreases in both c-Myc and IRF4 protein expression levels (70%-90% and 60%-80%, respectively) that were maximized by day 4. In turn, shRNA knockdown of either c-Myc or IRF4 elicited anti-proliferative (> 80% inhibition) and pro-apoptotic (50%-80%) responses as early as 48hrs after shRNA induction. These data suggest that the reduction of c-Myc and IRF4 protein levels downstream of Aiolos and Ikaros degradation account for the apoptotic effect and marks the onset of the cytotoxic response induced by LEN and POM in MM cells. To define the temporal order of events involving Aiolos, Ikaros, c-Myc and IRF4 in more detail, kinetic experiments following shRNA-mediated knockdown in parallel with drug treatments were performed. Data from these experiments showed that there is a distinct kinetic order of both LEN- and POM-mediated effects, initiated by immediate targeted degradation of Aiolos and Ikaros (within 90 min), followed by a decrease in c-Myc levels (24-48 hrs) with subsequent IRF4 downregulation (48-72 hrs), and finally, resulting in programmed cell death (3-5 days). Importantly, DOXY washout experiments, resulting in re-accumulation of Aiolos or Ikaros at early time points (24 hrs) partially overcame the antiproliferative effects of the shRNA-mediated knockdown of either target. Interestingly, upon the onset of c-Myc downregulation (24-48 hrs), the commitment to cell death could no longer be reversed in our experiments. Further, we generated MM1.S and U266 cells with acquired resistance to POM (10 µM; also cross-resistant to LEN) (MM1.S/PomR and U266/PomR , respectively), in which CRBN protein expression is substantially decreased (> 90%). Consequently, in these resistant cell lines, neither Aiolos nor Ikaros are degraded in the presence of LEN or POM. However, bypass of CRBN-dependent Aiolos degradation by DOXY-induced knockdown rescued c-Myc and IRF4 downregulation and concomitant inhibition of growth (90% and 60%, respectively), suggesting that resistant MM cells with acquired CRBN loss remain dependent on Aiolos and Ikaros. Conclusions: For the first time, our studies showed that degradation of Aiolos and Ikaros sets up a molecular sequence of events culminating in programmed cell death in MM cells. Our mechanistic studies showed that c-Myc is a key intermediate factor whose downregulation is a rate-limiting step for the transcriptional downregulation of IRF4 as well as for the commitment to cell death. Taken together, our results demonstrate a molecular sequence of events underlying the mechanism of action of cytotoxicity of LEN or POM in MM cells. Quantitative measurements of Aiolos and Ikaros degradation, and c-Myc and IRF4 downregulation in clinical samples would help validate these findings. Disclosures Bjorklund: Celgene Corp: Employment, Equity Ownership. Havens:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Amatangelo:Celgene Corp: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Wang:Celgene Corp: Consultancy. Breider:Celgene Corp: Employment. Ren:Celgene Corp: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

Sign in / Sign up

Export Citation Format

Share Document