scholarly journals Targeting PDZ-binding kinase is anti-tumorigenic in novel preclinical models of ACC

2019 ◽  
Vol 26 (10) ◽  
pp. 765-778 ◽  
Author(s):  
Adwitiya Kar ◽  
Yu Zhang ◽  
Betelehem W Yacob ◽  
Jordan Saeed ◽  
Kenneth D Tompkins ◽  
...  
Keyword(s):  
Cell Lines ◽  
Therapeutic Potential ◽  
Primary Therapy ◽  
Preclinical Models ◽  
H3 Phosphorylation ◽  
Promising Therapeutic Target ◽  
Wide Range ◽  
Advance Research ◽  
Induced Apoptosis

Adrenocortical carcinoma (ACC) is an aggressive orphan malignancy with less than 35% 5-year survival and 75% recurrence. Surgery remains the primary therapy and mitotane, an adrenolytic, is the only FDA-approved drug with wide-range toxicities and poor tolerability. There are no targeted agents available to date. For the last three decades, H295R cell line and its xenograft were the only available preclinical models. We recently developed two new ACC patient-derived xenograft mouse models and corresponding cell lines (CU-ACC1 and CU-ACC2) to advance research in the field. Here, we have utilized these novel models along with H295R cells to establish the mitotic PDZ-binding kinase (PBK) as a promising therapeutic target. PBK is overexpressed in ACC samples and correlates with poor survival. We show that PBK is regulated by FOXM1 and targeting PBK via shRNA decreased cell proliferation, clonogenicity and anchorage-independent growth in ACC cell lines. PBK silencing inhibited pAkt, pp38MAPK and pHistone H3 altering the cell cycle. Therapeutically, targeting PBK with the small-molecule inhibitor HITOPK032 phenocopied PBK-specific modulation of pAkt and pHistone H3, but also induced apoptosis via activation of JNK. Consistent with in vitro findings, treatment of CU-ACC1 PDXs with HITOPK032 significantly reduced tumor growth by 5-fold (P < 0.01). Treated tumor tissues demonstrated increased rates of apoptosis and JNK activation, with decreased pAkt and Histone H3 phosphorylation, consistent with effects observed in ACC cell lines. Together these studies elucidate the mechanism of PBK in ACC tumorigenesis and establish the potential therapeutic potential of HITOPK032 in ACC patients.

scholarly journals Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2012
Author(s):  
Kathryn M. Appleton ◽  
Charuta C. Palsuledesai ◽  
Sean A. Misek ◽  
Maja Blake ◽  
Joseph Zagorski ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Therapeutic Potential ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Intrinsic Resistance ◽  
Primary Resistance ◽  
Induced Apoptosis ◽  
Melanoma Cell Lines

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAFV600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

scholarly journals Therapeutic potential of SGN-CD19B, a PBD-based anti-CD19 drug conjugate, for treatment of B-cell malignancies

Blood ◽  
2017 ◽  
Vol 130 (18) ◽  
pp. 2018-2026 ◽  
Author(s):  
Maureen C. Ryan ◽  
Maria Corinna Palanca-Wessels ◽  
Brian Schimpf ◽  
Kristine A. Gordon ◽  
Heather Kostner ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Preclinical Models ◽  
B Cell Malignancies ◽  
Key Points

Key Points SGN-CD19B is broadly active in vitro against malignant B-cell lines, including double-hit and triple-hit lymphoma cell lines. SGN-CD19B shows significant antitumor activity in vivo in preclinical models of B-NHL and B-cell–derived acute lymphoblastic leukemia.

Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2797-2805 ◽  
Author(s):  
Feng-Ting Liu ◽  
Samir G. Agrawal ◽  
John G. Gribben ◽  
Hongtao Ye ◽  
Ming-Qing Du ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Resistant Cell ◽  
Protein Levels ◽  
Bax Protein ◽  
Degradation Activity ◽  
Potent Drug ◽  
Induced Apoptosis

Proapoptotic Bcl-2 family member Bax is a crucial protein in the induction of apoptosis, and its activation is required for this process. Here we report that Bax is a short-lived protein in malignant B cells and Bax protein levels decreased rapidly when protein synthesis was blocked. Malignant B cells were relatively resistant to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–induced apoptosis, and this correlated with low basal Bax protein levels. Furthermore, during treatment with TRAIL, the resistant cell lines showed prominent Bax degradation activity. This degradation activity was localized to mitochondrial Bax and could be prevented by truncated Bid, a BH3-only protein; in contrast, cytosolic Bax was relatively stable. The proteasome inhibitor bortezomib is a potent drug in inducing apoptosis in vitro in malignant B-cell lines and primary chronic lymphocytic leukemic (CLL) cells. In CLL cells, bortezomib induced Bax accumulation, translocation to mitochondria, conformational change, and oligomerization. Accumulation and stabilization of Bax protein by bortezomib-sensitized malignant B cells to TRAIL-induced apoptosis. This study reveals that Bax instability confers resistance to TRAIL, which can be reversed by Bax stabilization with a proteasome inhibitor.

Targeted chemical nucleases have a wide range of untapped applications in biological fields, including gene therapy

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Metal Complex ◽  
Dna Cleavage ◽  
Therapeutic Potential ◽  
Chemical Reactivity ◽  
Major Groove ◽  
Sequence Recognition ◽  
Wide Range ◽  
Chemical Nucleases

A feasible alternative to state-of-the-art enzymatic nucleases was created by regulating the cleavage activity of metal complexes using (covalent or non-covalent) homing agents. Targeted AMNs, unlike enzymatic nucleases, break DNA by an oxidative mechanism and can therefore permanently knock off genes. Compared to larger enzymatic nucleases, the modest size of the metal complex may aid cellular transfection. Furthermore, the painstaking construction of the sequence-specific probe permits a metal complex to be directed to dsDNA's minor or major groove. To direct the chemical reactivity of several small-molecule compounds to dsDNA's minor groove, covalently bonded polyamide samples were used. PNA and DNA were also used to construct antisense and antigen hybrids, with Watson–Crick or Hoogsteen base pairing with major groove nucleobases giving sequence recognition. Click chemistry created chimeric AMN-TFOs with desirable focused effects and negligible off-target cleavage. Clip-Phen-modified TFOs, 230 polypyridyl-modified TFOs, 232 and intercalating phenanthrene-modified TFOs are three contemporary instances of copper AMN–TFOs. All three systems have distinct advantages in maintaining the desired 2:1 phenthroline/copper ratio for DNA cleavage (clip-Phen TFOs), caging the copper center and facilitating efficient ROS-mediated strand scission (polypyridyl-modified TFO) and improving triplex stability (polypyridyl-modified TFO) (phenanthrene-TFOs). Cerium (IV)/EDTA complexes, recently shown to bind and hydrolytically cleave ssDNA/dsDNA junctions and used in conjunction with PNA to successfully introduce genome changes in vitro and in vivo, are another important class of targeted chemical nucleases. The chemical reactivity and wide flexibility of metal complex design, combined with their coupling to sequence specific samples for directed applications, show that these compounds have a wide range of untapped applications in biological fields such as chemotherapy, protein engineering, DNA footprinting, and gene editing. Parallel advancements in cell and tissue targeting will be essential to maximise their therapeutic potential, either by using specific ligands or creating new targeting modalities.

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes

1983 ◽  
Vol 3 (4) ◽  
pp. 596-604
Author(s):  
C A Whitlock ◽  
S F Ziegler ◽  
O N Witte
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Lymphoid Cells ◽  
Growth Potential ◽  
Malignant Growth ◽  
Wide Range ◽  
Feeder Layers ◽  
Growth Phenotype ◽  
Abelson Virus

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.

Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic β-cells

2008 ◽  
Vol 294 (3) ◽  
pp. E540-E550 ◽  
Author(s):  
Elida Lai ◽  
George Bikopoulos ◽  
Michael B. Wheeler ◽  
Maria Rozakis-Adcock ◽  
Allen Volchuk
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Human Islets ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Min6 Cells ◽  
Relative Contribution ◽  
Induced Apoptosis

Chronic exposure to elevated saturated free fatty acid (FFA) levels has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic β-cell apoptosis. Here, we compared the effects of FFAs on apoptosis and ER stress in human islets and two pancreatic β-cell lines, rat INS-1 and mouse MIN6 cells. Isolated human islets cultured in vitro underwent apoptosis, and markers of ER stress pathways were elevated by chronic palmitate exposure. Palmitate also induced apoptosis in MIN6 and INS-1 cells, although the former were more resistant to both apoptosis and ER stress. MIN6 cells were found to express significantly higher levels of ER chaperone proteins than INS-1 cells, which likely accounts for the ER stress resistance. We attempted to determine the relative contribution that ER stress plays in palmitate-induced β-cell apoptosis. Although overexpressing GRP78 in INS-1 cells partially reduced susceptibility to thapsigargin, this failed to reduce palmitate-induced ER stress or apoptosis. In INS-1 cells, palmitate induced apoptosis at concentrations that did not result in significant ER stress. Finally, MIN6 cells depleted of GRP78 were more susceptible to tunicamycin-induced apoptosis but not to palmitate-induced apoptosis compared with control cells. These results suggest that ER stress is likely not the main mechanism involved in palmitate-induced apoptosis in β-cell lines. Human islets and MIN6 cells were found to express high levels of stearoyl-CoA desaturase-1 compared with INS-1 cells, which may account for the decreased susceptibility of these cells to the cytotoxic effects of palmitate.

scholarly journals Clinical Pharmacodynamic Markers and Combinations with SY1425 (tamibarotene) in a Genomically-Defined Subset of Non-APL AML

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2898-2898
Author(s):  
Michael R McKeown ◽  
Christopher Fiore ◽  
Emily Lee ◽  
Matthew L Eaton ◽  
Christian C. Fritz
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Binding Sites ◽  
Clinical Studies ◽  
High Cell ◽  
Preclinical Models ◽  
Employment Equity ◽  
Equity Ownership ◽  
Maximal Induction

Abstract SY-1425, a potent and selective agonist of the retinoic acid receptor RARα, is being investigated in a Ph2 trial in a novel genomically-defined subset of non-APL AML and MDS patients (clinicaltrials.gov NCT02807558). RARa is a nuclear hormone receptor and transcription factor that regulates genes involved in cell differentiation and proliferation. We identified a super-enhancer (SE) at the RARA locus, the gene encoding RARa, in a subset of primary non-APL AML blasts. Preclinical models demonstrated a correlation between the presence of a RARA SE and sensitivity to SY-1425, providing the rationale for clinical investigation. Further research has investigated pharmacodynamics (PD) markers and combinations of drugs to support clinical development of SY-1425. In this study we identified DHRS3mRNA induction as a measure of RARα target engagement with SY-1425. We also demonstrated synergy in preclinical models with SY-1425 and hypomethylating agents. Since RARα is a transcription factor that regulates target genes when bound by a retinoid, we characterized the dynamic expression changes of a panel of RARA enhancer- high and - low non-APL AML cell lines (hereafter referred to as RARA-high and -low) in response to SY-1425 treatment. DHRS3 showed the largest expression increase following treatment in 3 RARA-high cell lines, with a range of 29 to 115 fold. In contrast, there was a much lower DHRS3 induction in 3 RARA-low cell lines (range of 1.6 to 6.1 fold). Induction was found to be both time- and dose-dependent with maximal induction at approximately 6 hours and half maximal induction near the EC50 for the anti-proliferative effect in RARA-high cell lines. DHRS3 encodes dehydrogenase/reductase (SDR family) member 3, a metabolic enzyme involved in maintaining cellular retinol homeostasis and had previously been shown to be induced by retinoids. Thus, DHRS3induction in tumor cells represents a potentially useful PD marker for clinical studies of SY-1425. To better understand the mechanism of induction of DHRS3 by SY-1425 we examined the chromosomal localization of RARα as well as the epigenomic state of the DHRS3 locus by ChIP-seq for RARα and H3K27 acetylation, the latter being an indicator of active enhancers and promoters. In the untreated state, OCI-AML3 (a typical RARA-high AML cell line) was found to have multiple RARα binding sites both within and distal to the DHRS3 gene but minimal H3K27 acetylation. Following treatment with SY-1425, the level of H3K27 acetylation at DHRS3 increased, resulting in the formation of a SE. Moreover, the SE encompassed the RARα binding sites, consistent with the model in which SY-1425 converts RARα into an activator of DHRS3expression. Similar results were seen for the CD38 locus in which SY-1425 treatment increased expression, H3K27 acetylation, and RARα binding. CD38 is a cell surface antigen and marker of myeloid maturation readily analyzed by FACS analysis, suggesting it could be an additional PD marker to be used in clinical studies. Indeed, it was found that SY-1425 induced CD38 cell surface expression at similar levels in RARA-high AML cell lines and the NB-4 APL cell line, but not in RARA-low cell lines. We also investigated combinations of SY-1425 with approved or investigational AML and MDS agents in in vitro and in vivo models to inform future clinical studies and to further explore potential PD markers unique to the combined action of the drugs. Several standard of care agents and drugs in current development were found to have synergistic interactions with SY-1425 in RARA-high but not RARA-low cell lines. In particular, azacitidine and decitabine each showed strong in vitro synergy with SY-1425. Evaluation of SY-1425 plus azacitidine in a RARA-high PDX model of non-APL AML demonstrated a better response compared to either agent alone. Additional genome-wide ChIP-seq and expression studies of RARA-high cells treated with various combinations are being investigated to identify optimal PD markers for these combinations. These studies support the use of DHRS3 mRNA induction in tumor cells as a PD marker in the recently initiated Ph2 study of SY-1425 in genomically-defined non-APL AML and MDS patients (clinicaltrials.gov NCT02807558) and further exploration as a PD marker for future combination studies. Disclosures McKeown: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

scholarly journals Quantitative analysis of tumour spheroid structure

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alexander P Browning ◽  
Jesse A Sharp ◽  
Ryan J Murphy ◽  
Gency Gunasingh ◽  
Brodie Lawson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tumour Microenvironment ◽  
Culture Conditions ◽  
Experimental Models ◽  
Seeding Density ◽  
Modelling Framework ◽  
Tumour Spheroid ◽  
Tumour Spheroids ◽  
Wide Range

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

Blood ◽  
2021 ◽  
Author(s):  
Roger Belizaire ◽  
Sebastian Hassan John Koochaki ◽  
Namrata D. Udeshi ◽  
Alexis Vedder ◽  
Lei Sun ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Therapeutic Potential ◽  
Mutant Cell ◽  
E3 Ubiquitin Ligase ◽  
Akt Signaling ◽  
Allelic Series ◽  
Genetic Ablation

CBL encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and non-receptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10-20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, PIK3R1 recruitment, and downstream PI3K/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine-kinase binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

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