Pre-clinical use of isogenic cell lines and tumours in vitro and in vivo for predictive biomarker discovery; impact of KRAS and PI3KCA mutation status on MEK inhibitor activity is model dependent

ABSTRACTDespite the anticancer activity of pan-histone deacetylase (HDAC) inhibitors, their clinical use has been limited due to toxicity. However, the development of more specific inhibitors that selectively inhibit individual HDACs is emerging as a novel and well-tolerated alternative. Here, we present the results of the first clinical trial evaluating the activity of ricolinostat (the leading HDAC6 inhibitor) in breast cancer (BC) patients.We have developed a computational network-based algorithm to evaluate the activity of the HDAC6 protein, based on the enrichment of its transcriptional targets in differentially expressed genes (HDAC6 score). Through preclinical in vitro and in vivo studies, we confirmed that the HDAC6 score can stratify the sensitivity of BC cells to ricolinostat treatment and may thus have value as a predictive biomarker. Moreover, analysis of ∼3,000 primary human breast cancers showed that ∼30% of them present high HDAC6 scores. Based on these results, we designed a phase Ib clinical trial to evaluate the activity of ricolinostat plus nab-paclitaxel in metastatic BC patients. Study results showed that the two agents can be safely combined, that clinical activity is identified specifically in patients with HR+/HER2-disease, and that the HDAC6 score was predictive of response. Expansion of our analysis to other tumor types identified multiple cohorts enriched in high HDAC6 score samples. These results suggest that the HDAC6 score may provide an effective, CLIA certified predictive biomarker of ricolinostat sensitivity in multiple human cancers.SIGNIFICANCEThe clinical use of HDAC inhibitors is hampered by the toxicity associated with blocking multiple HDACs. Here, we show that the specific HDAC6 inhibitor ricolinostat is safe and presents clinical activity in breast cancers and that the HDAC6 score has predictive biomarker potential to identify patients who can benefit from this therapy.

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Pathway profiling of a novel SRC inhibitor, AZD0424, in combination with MEK inhibitors

10.1101/2021.08.27.457893 ◽

2021 ◽

Author(s):

John C Dawson ◽

Alison Munro ◽

Kenneth Macleod ◽

Morwenna Muir ◽

Paul Timpson ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Cancer Cell ◽

Mek Inhibitor ◽

Cancer Cell Lines ◽

Drug Induced ◽

Combination Strategies ◽

Src Inhibitor

AbstractA more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments allows us anticipate how cells adapt to targeted therapy enabling more informed prediction of rational drug combinations. The non-receptor tyrosine kinase SRC regulates many cellular signalling processes and pharmacological inhibition has long been a target of drug discovery projects for the treatment of cancer. Here we describe the in vitro and in vivo characterisation of the small molecule SRC inhibitor, AZD0424. We show that AZD0424 potently inhibits the phosphorylation of tyrosine-416 of SRC (IC50 ∼ 100 nM) in many cancer cell lines; however inhibition of cell viability, via a G1 cell cycle arrest, was observed only in a sub-set of cancer cell lines in the low (on target) micromolar range. We profiled the changes in intracellular pathway signalling in cancer cells following exposure to AZD0424 and other targeted therapies using Reverse Phase Protein Array analysis. We demonstrate that SRC is activated in response to MEK inhibitor (trametinib or AZD6244)-treatment of KRAS mutant colorectal cell lines (HCT116 and DLD1) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 revealed reduction of EGFR, FAK and SRC compensatory activation, and, synergistically inhibits cell viability in vitro. In vivo, trametinib-treatment of mice bearing HCT116 tumours increased phosphorylation of SRC on Tyr416, and when combined with AZD0424, inhibition of tumour growth is greater than trametinib alone. We also demonstrate that drug-induced resistance to trametinib is not re-sensitised by AZD0424 treatment in vitro, likely as a result of multiple compensatory signalling mechanisms; however inhibition of SRC remains an effective way to block invasion of trametinib resistant tumour cells. These data imply that inhibiting SRC may offer a useful addition to MEK inhibitor combination strategies.

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ALKBH5 Functions As an Oncogene in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-118988 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3910-3910

Author(s):

Chao Shen ◽

Yue Sheng ◽

Rui Su ◽

Xiaolan Deng ◽

Sean Robinson ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Myeloid Leukemia ◽

Tp53 Mutation ◽

Mouse Bone Marrow ◽

Mutation Status ◽

Acute Myeloid

Abstract N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic messenger RNAs (mRNAs) has been shown to play important roles in diverse cellular and pathological processes (Deng X, et al. Cell Res. 2018;28:507-517). ALKBH5, recently identified as a m6A demethylase, was reported to promote tumorigenesis and proliferation in glioblastoma stem-like cells (GSCs) (Zhang, S. et al. Cancer Cell. 2017;31:591-606) and breast cancer stem cells (BCSCs) (Zhang, C et al. PNAS. 2016;113: E2047-E2056). While ALKBH5 is well-recognized to function as an oncogene in solid tumors, it was reported that shallow/deep deletion of ALKBH5 is associated poor prognosis in patients with acute myeloid leukemia (AML), and is frequently co-existing with TP53 mutation (Kwok, C. T et al. J Hematol Oncol. 2017; 10(1): 39), implying that ALKBH5 may function as a tumor suppressor in AML. Thus, a systematic investigation of the definitive role of ALKBH5 in AML is warranted. To this end, we performed series of in vitro and in vivo experiments to determine the function of ALKBH5 in AML. For the in vitro experiments, we used three lentiviral shRNAs (shALKBH5-A, shALKBH5-D and shALKBH5-E) to deplete ALKBH5 expression in three human AML cell lines with different TP53 mutation status: NOMO-1 (TP53-mutant), MV4;11 (TP53-WT) and MA9.3 cells (TP53-WT). Somewhat surprisingly, ALKBH5 depletion significantly (p<0.05) inhibited AML cell proliferation/growth in all three AML cells lines, regardless of the status of TP53 mutation. We next conducted colony forming assays and found that ALKBH5 knockdown significantly (p<0.01) impaired the colony forming ability to 18% ~45% of the control group level in all three AML cell lines. We further showed that ALKBH5 depletion caused a significant increase in apoptosis (with a 1.5 ~ 4 fold increase; p<0.001) in all three AML cell lines, which is consistent with the previous report that knockout of ALKBH5 caused severe apoptosis of mouse testis cells (Zheng G et al. Mol Cell. 2013; 49:18-29). In contrast, ALKBH5 knockdown did not significantly affect cell cycles. To further confirm ALKBH5's role in AML development in vivo. We utilized Xenografted AML model as well as mouse bone marrow transplantation (BMT) model. Consistent with the in vitro results, we found that NSGS mice xeno-transplanted with MV4;11-ALKBH5-knockdown cells survived significantly longer than those with MV4;11 control cells (p<0.001). Moreover, we have also conducted mouse bone marrow transplantation (BMT) assays with MLL-AF9-transduced mouse bone marrow lineage negative (Lin-) progenitor cells collected from mice carrying Alkbh5 wild-type (Alkbh5+/+), or heterozygous (Alkbh5+/-) or homozygous (Alkbh5-/-) deletion. Consistent with the xeno-transplanted mouse model results, our BMT assays also showed that Alkbh5 depletion significantly inhibited leukemogenesis and prolonged survival in BMT recipient mice (median survival of ALKBH5wt/wt +MA9 vs. ALKBH5+/- +MA9 or ALKBH5-/- +MA9: 32 days vs. 64 days or 68 days; p<0.005). Taken together, our in vitro and in vivo functional studies data indicate ALKBH5 also functions as an oncogene in AML regardless of TP53 mutation status, similar to its role in solid tumors. We are currently conducting as series of studies to reveal the molecular mechanism(s) underlying the oncogenic role of ALKBH5 in AML. Disclosures No relevant conflicts of interest to declare.

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A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650089 ◽

1980 ◽

Vol 44 (02) ◽

pp. 081-086 ◽

Cited By ~ 8

Author(s):

C V Prowse ◽

A E Williams

Keyword(s):

Platelet Rich Plasma ◽

Thrombus Formation ◽

Factor Ix ◽

Coagulation System ◽

In Vitro Tests ◽

Clinical Use ◽

Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

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Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655587 ◽

1964 ◽

Vol 12 (01) ◽

pp. 232-261 ◽

Cited By ~ 2

Author(s):

S Sasaki ◽

T Takemoto ◽

S Oka

Keyword(s):

Molecular Weight ◽

Dextran Sulfate ◽

Anticoagulant Activity ◽

Molecular Structures ◽

Severe Reaction ◽

Clinical Use ◽

Molecular Weights ◽

Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

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Inhibition of Fibrinolysis and Fibrinogenolysis in Man: Comparison of ε-Aminocaproic Acid and Kallikrein Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660337 ◽

1963 ◽

Vol 10 (01) ◽

pp. 106-119 ◽

Cited By ~ 8

Author(s):

E Beck ◽

R Schmutzler ◽

F Duckert ◽

Keyword(s):

Aminocaproic Acid ◽

Side Reactions ◽

In Vitro Tests ◽

Factor V ◽

Clinical Use ◽

Strong Inhibitor ◽

Kallikrein Inhibitor ◽

Therapeutic Possibilities

SummaryInhibitor of kallikrein and trypsin (KI) extracted from bovine parotis was compared with ε-aminocaproic acid (EACA): both substances inhibit fibrinolysis induced with streptokinase. EACA is a strong inhibitor of fibrinolysis in concentrations higher than 0, 1 mg per ml plasma. The same amount and higher concentrations are not able to inhibit completely the proteolytic-side reactions of fibrinolysis (fibrinogenolysis, diminution of factor V, rise of fibrin-polymerization-inhibitors). KI inhibits well proteolysis of plasma components in concentrations higher than 2,5 units per ml plasma. Much higher amounts of KI are needed to inhibit fibrinolysis as demonstrated by our in vivo and in vitro tests.Combination of the two substances for clinical use is suggested. Therapeutic possibilities are discussed.

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LPTO-06. A NOVEL BRAIN-PERMEANT CHEMOTHERAPEUTIC AGENT FOR THE TREATMENT OF BREAST CANCER LEPTOMENINGEAL METASTASIS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdz014.029 ◽

2019 ◽

Vol 1 (Supplement_1) ◽

pp. i7-i7

Author(s):

Jiaojiao Deng ◽

Sophia Chernikova ◽

Wolf-Nicolas Fischer ◽

Kerry Koller ◽

Bernd Jandeleit ◽

...

Keyword(s):

Breast Cancer ◽

Brain Tumors ◽

Cell Lines ◽

Chemotherapeutic Agent ◽

Leptomeningeal Metastasis ◽

Breast Cancer Cell Lines ◽

Normal Brain ◽

Breast Cancers

Abstract Leptomeningeal metastasis (LM), a spread of cancer to the cerebrospinal fluid and meninges, is universally and rapidly fatal due to poor detection and no effective treatment. Breast cancers account for a majority of LMs from solid tumors, with triple-negative breast cancers (TNBCs) having the highest propensity to metastasize to LM. The treatment of LM is challenged by poor drug penetration into CNS and high neurotoxicity. Therefore, there is an urgent need for new modalities and targeted therapies able to overcome the limitations of current treatment options. Quadriga has discovered a novel, brain-permeant chemotherapeutic agent that is currently in development as a potential treatment for glioblastoma (GBM). The compound is active in suppressing the growth of GBM tumor cell lines implanted into the brain. Radiolabel distribution studies have shown significant tumor accumulation in intracranial brain tumors while sparing the adjacent normal brain tissue. Recently, we have demonstrated dose-dependent in vitro and in vivo anti-tumor activity with various breast cancer cell lines including the human TNBC cell line MDA-MB-231. To evaluate the in vivo antitumor activity of the compound on LM, we used the mouse model of LM based on the internal carotid injection of luciferase-expressing MDA-MB-231-BR3 cells. Once the bioluminescence signal intensity from the metastatic spread reached (0.2 - 0.5) x 106 photons/sec, mice were dosed i.p. twice a week with either 4 or 8 mg/kg for nine weeks. Tumor growth was monitored by bioluminescence. The compound was well tolerated and caused a significant delay in metastatic growth resulting in significant extension of survival. Tumors regressed completely in ~ 28 % of treated animals. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, Quadriga’s new agent could be effective as a therapeutic for both primary and metastatic brain tumors such as LM. REF: https://onlinelibrary.wiley.com/doi/full/10.1002/pro6.43

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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Delivery of melittin-loaded niosomes for breast cancer treatment: an in vitro and in vivo evaluation of anti-cancer effect

Cancer Nanotechnology ◽

10.1186/s12645-021-00085-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Farnaz Dabbagh Moghaddam ◽

Iman Akbarzadeh ◽

Ehsan Marzbankia ◽

Mahsa Farid ◽

Leila khaledi ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Treatment ◽

Cell Lines ◽

Encapsulation Efficiency ◽

Inbred Mice ◽

Breast Cancer Treatment ◽

Anticancer Effect ◽

Anti Cancer

Abstract Background Melittin, a peptide component of honey bee venom, is an appealing candidate for cancer therapy. In the current study, melittin, melittin-loaded niosome, and empty niosome had been optimized and the anticancer effect assessed in vitro on 4T1 and SKBR3 breast cell lines and in vivo on BALB/C inbred mice. "Thin-layer hydration method" was used for preparing the niosomes; different niosomal formulations of melittin were prepared and characterized in terms of morphology, size, polydispersity index, encapsulation efficiency, release kinetics, and stability. A niosome was formulated and loaded with melittin as a promising drug carrier system for chemotherapy of the breast cancer cells. Hemolysis, apoptosis, cell cytotoxicity, invasion and migration of selected concentrations of melittin, and melittin-loaded niosome were evaluated on 4T1 and SKBR3 cells using hemolytic activity assay, flow cytometry, MTT assay, soft agar colony assay, and wound healing assay. Real-time PCR was used to determine the gene expression. 40 BALB/c inbred mice were used; then, the histopathology, P53 immunohistochemical assay and estimate of renal and liver enzyme activity for all groups had been done. Results This study showed melittin-loaded niosome is an excellent substitute in breast cancer treatment due to enhanced targeting, encapsulation efficiency, PDI, and release rate and shows a high anticancer effect on cell lines. The melittin-loaded niosome affects the genes expression by studied cells were higher than other samples; down-regulates the expression of Bcl2, MMP2, and MMP9 genes while they up-regulate the expression of Bax, Caspase3 and Caspase9 genes. They have also enhanced the apoptosis rate and inhibited cell migration, invasion in both cell lines compared to the melittin samples. Results of histopathology showed reduce mitosis index, invasion and pleomorphism in melittin-loaded niosome. Renal and hepatic biomarker activity did not significantly differ in melittin-loaded niosome and melittin compared to healthy control. In immunohistochemistry, P53 expression did not show a significant change in all groups. Conclusions Our study successfully declares that melittin-loaded niosome had more anti-cancer effects than free melittin. This project has demonstrated that niosomes are suitable vesicle carriers for melittin, compare to the free form.

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Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

Scientific Reports ◽

10.1038/s41598-021-87700-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mary Jo Rademacher ◽

Anahi Cruz ◽

Mary Faber ◽

Robyn A. A. Oldham ◽

Dandan Wang ◽

...

Keyword(s):

Immune Response ◽

Cell Lines ◽

Nk Cell ◽

Autologous Tumor ◽

Murine Models ◽

Lentiviral Transduction ◽

Ifn Γ ◽

Human Sarcoma

AbstractInterleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

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