The Thermosensitizing Effects of Kinase
Inhibitors on Cervical Cancer Cell Cines
By Ingmar Plijter
11650788
BSc Biomedical Sciences
5-month internship (February 2020 – June 2020) 3636 words
Daily Mentor: Senior Investigator:
E.M. Scutigliani P.M. Krawczyk
1
ABSTRACT
Nowadays, the main conventional methods to combat cancer are radiotherapy, chemotherapy, and surgery, however, these treatments all have their limitations, increasing the demand for a more efficient method for cancer treatment. A treatment method that is rapidly gaining popularity is hyperthermia treatment, however, much remains to be investigated to optimize this treatment method. Previous research found that protein kinases are often mutated in cancer, and are associated with tumor progression because they are key regulators of cell growth and cell division. It has never been systematically investigated how kinase inhibitor drugs could potentially improve the effects of hyperthermia treatment. This study therefore aimed to systematically evaluate the combinatorial effects of a wide variety of kinase inhibitor drugs and hyperthermia. To investigate this, a high-throughput drug screen was performed using more than 1400 kinase inhibitor drugs, of which the top ten were validated. The drugs validated were Pomalidomide, SGI-1027, Aminophylline, NSC319726, UNC0631, Nebivolol, Silodosin, Zalcitabine, Dasatinib, and Ketoprofen. It was found that the sensitizing effect of the kinase inhibitor drugs in hyperthermia treatment is not related to one specific cellular process or pathway, furthermore, it has been found that G9a/GLP inhibition by UNC0631 and Pomalidomide, an immunomodulatory antineoplastic agent, can be a potential target for improving hyperthermia treatment. Further research should investigate the mechanistic effect of UNC0631 and Pomalidomide to improve hyperthermia treatment.
INTRODUCTION
Cancer is a major public health problem worldwide. It is expected that cancer will rank as the global leading cause of death due to the rising incidence and mortality rates and will therefore become the biggest barrier to extending life expectancy in the 21st century (Bray et al., 2018). As the incidence of
cancer is expected to increase as a result of the growing population and age, it is important that the scientific community addresses this problem (Torre et al., 2016). In recent years, a lot of progress has been made in the understanding of cancer. Several hallmarks have been found, such as evasion of apoptosis, the induction of angiogenesis, and the capability to invade or metastasize (Hanahan & Weinberg, 2011). Due to the increased knowledge in the past decade, enormous steps have been taken in the diagnosis and treatments of many cancer types (Baskar et al., 2012). Nowadays, the main conventional methods to combat cancer are radiotherapy, chemotherapy, and surgery. These treatments all have their limitations since not all tumors are operable, and the addition of chemo- and radiotherapy is limited to a maximum dose to protect the surrounding tissue (Chen & Kuo, 2017). These limitations therefore increase the demand for novel treatment strategies (Hehr et al., 2003). A treatment method that is rapidly gaining popularity is hyperthermia treatment, which has already been implemented in cancer therapy as an adjuvant to chemo- and radiotherapy, in the treatment of colorectal cancer, bladder cancer, prostate cancer, melanoma, cervical cancer, peritoneal carcinomas, and head and neck cancers (Hehr et al., 2003; Rao & Deng, 2010).
There are currently two types of hyperthermia treatments: thermal ablation therapy and mild hyperthermia. In thermal ablation therapy, cells are heated to a minimum of 45°C, causing immediate cell death. In mild hyperthermia (41-43°C), the cancerous tissue is mainly heated to make the tissue more susceptible to the conventional treatments (Hehr et al., 2003). A further distinction is made between whole-body-, regional- and local hyperthermia treatment. Whole-body hyperthermia is when the whole body is heated, compared to the regional treatment where only a part of the body is exposed to heat. Local hyperthermia treatment focuses on heating the tumor tissue. This paper focuses on mild hyperthermia treatment.
2 Treatment with mild hyperthermia has been found to enhance the effects of conventional treatments. Chemotherapy is enhanced by hyperthermia due to increased blood flow resulting in vascular leakage, which facilitates the uptake of anti-cancer drugs (e.g. chemotherapeutics) into the tumor to enhance the cytotoxic effect on the tumor. Radiotherapy, on the other hand, is strengthened because the DNA damage caused by radicals cannot be repaired (Jha et al., 2016). The hyperthermia treatment influences many cellular processes. It was found that hyperthermia causes problems in DNA repair and overall protein functioning, leading to problems in all cell organelles. Some effects of hyperthermia may have added value in the treatment of cancer. Van den Tempel (2016) has already written a detailed review of how hyperthermia can be used in cancer treatment.
Hyperthermia is thus combined with chemo- and radiotherapy in the clinic. No studies, however, have yet attempted to systematically examine whether interfering with other cellular processes in cancer cells could strengthen the effects of hyperthermia. Protein kinases are often mutated and are associated with tumor progression because they are the key regulators of cell growth and cell division but they also play a role in many other cellular processes (Bhullar et al., 2018). By inhibiting a certain cellular process, the hyperthermia treatment could be potentially enhanced. This study therefore investigates how the desired effects of hyperthermia treatment in human cervical cancer can be enhanced by using a kinase inhibitor drug, since hyperthermia treatment is currently routinely used in the treatment of cervical cancer (Kroesen et al., 2019). To investigate this, a high-throughput drug screen was performed, by members of our group. More than 1400 kinase inhibitor drugs were tested to see which drugs enhance the hyperthermia effect. The most promising drugs were selected and validated. In this study, it has been found that G9a/GLP inhibition by UNC0631 and the use of a Pomalidomide, an immunomodulatory antineoplastic agent, can be an interesting target for improving hyperthermia treatment.
MATERIALS AND METHODS
Culture of cancer cells
The human cervical cancer cell lines HeLa and SiHa were obtained from the American Type Culture Collection (ATCC). The cells were cultured in Eagle’s minimal essential medium (EMEM, Lonza), which was supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin (100 µg/ml, Gibco), 1% Streptomycin (100 U/ml, Gibco) and L-Glutamine (292 µg/ml, Gibco). The cells were cultured at 37°C in a 5% CO2 humidified chamber.
Preliminary study
Cells were seeded 24 hours prior to hyperthermia in flat-bottom 96-well plates (Corning) at a density of 1000 cells/well. 1 hour before hyperthermia treatment, pre-diluted drugs from the kinase inhibitor library (Selleckchem) were added at a final concentration of 1 µM. Hyperthermia was subsequently performed as described in the next section. Cell viability was assessed 72 hours after hyperthermia by incubation with PrestoBlue™ Cell Viability Reagent (ThermoFisher) as described by the manufacturer, followed by quantification with a CLARIOstar® microplate reader (BMG LABTECH), by using an excitation wavelength of 560 ± 15 nm, a dichroic mirror set at 573.8 nm, and an detection wavelength of 590 ± 20 nm. After correcting for background noise, the cell viability was normalized to DMSO-treated controls, and the thermal enhancement ratio (TER) was calculated using the formula: ‘’viability drug alone” / “viability hyperthermia + drug’’. All treatments were performed in duplo.
3
Treatment modalities
The drug treatment was performed by seeding 4000 cells/well in triplicate, in a 24 well plate (Greiner), at a volume of 500 µl per well. Cells were incubated for 24 hours after which the drug treatment took place. All drugs were administered with fresh EMEM medium in four concentrations of 1 µM, 0.5 µM, 0.25 µM and 0 µM for both SiHa and HeLa cells, with a total volume of 500 µl per well.
Hyperthermia was conducted 30 minutes after drug administration, by submerging the culture plates in a thermostatically controlled water bath maintained at 42 °C and 5% CO2 for 1 hour and 5 minutes,
to achieve a clinically relevant condition of 42 °C for 1 hour.
After the hyperthermia treatment, the drugs were removed according to the schedule, based on incubation times relevant to the clinic (table 1). Incubation times were determined based on the half-life of the drug (Drugbank). The medium was replaced, and cells were incubated.
PrestoBlue survival assays
Five days after the hyperthermia treatment the PrestoBlue™ Cell Viability Assay was conducted. The EMEM medium was aspirated from the 24 well plates (Geigner) and replaced with a solution of 250 µl EMEM medium and PrestoBlue Cell™ Viability Reagent (ThermoFisher). Cells were then incubated for 90 minutes using a humidifier containing an atmosphere of 5% CO2. A CLARIOstar® High Performance
Monochromator Multimode Microplate Reader (BMG LABTECH) was then used to measure the fluorescence intensity using an emission wavelength of 590 nm ( ± 20 nm), a dichroic mirror for 573.8 nm and an excitation wavelength of 560 nm (± 15 nm).
Statistical analysis
Data from the groups which did or did not undergo hyperthermia treatment were independently normalized in Excel. Background noise was corrected, and cell viability was normalized to DMSO-treated controls. The data of the experimental group and control group was then compared and analyzed in GraphPad Prism 8 and tested for significance (P <0.05) using a student's t-test.
Table 1: The different incubations times and half-life of the drugs used
Drug Incubation time (h) Half-life (h) Silodosin 48 13.3 ± 8.07 Nebivolol 48 12 Pomalidomide 48 9.4 Aminophylline 48 7 – 9 SGI-1027 48 Unknown NSC319726 48 Unknown UNC0631 48 Unknown Dasatinib 24 3-5 Zalcitabine 24 2 Ketoprofen 24 1.1 – 4
4
RESULTS
1. The sensitizing effect of the kinase inhibitor drugs on hyperthermia treatment is not related to one specific cellular process or pathway.
The preliminary study showed that different kinase inhibitors can act as protector or sensitizer (figure
1). Top hits were chosen based on two factors, the TER, and whether the drugs are already FDA
approved (table 2). The mechanism of action of all top hits was subsequently globally examined, to see whether the drug's sensitizing effect in combination with hyperthermia affected a specific cellular process or pathway (figure 1C). The results showed that the sensitizing effect in SiHa cells is primarily mediated by kinase inhibitor drugs that play a role in DNA damage, followed by cytoskeletal signaling. In contrast, the sensitizing effect in HeLa cells is caused by kinase inhibitor acting on microbiology, followed by JAK/STAT pathway (figure 1C).
HeLa and SiHa cells combined show that the sensitizing effects are mainly mediated by drugs that interact with microbiology, endocrinology & hormones, JAK/STAT, and Protein Tyrosine kinases (figure 1C). This may indicate that some pathways or cellular processes are more responsible for the combinatorial effect, which may be worth investigating further. It does, however, show that sensitizing effects of the kinase inhibitor drug on hyperthermia are not related to one specific cellular process or pathway.
2. Pomalidomide, Dasatinib, Silodosin SGI-1027, NSC319726, and UNC0631 sensitize cervical cancer cells to hyperthermia.
The most promising drugs were selected based on the thermal enhancement ratio (TER) or whether the drug was already FDA approved. Pomalidomide is an FDA approved drug, which is an analog of thalidomide. Pomalidomide acts as an immunomodulatory antineoplastic agent, which shows antiangiogenic properties and is used in the treatment of multiple myeloma (Engelhardt et al., 2014). Dasatinib is another FDA approved cancer drug, which is used in the treatment of chronic myeloid leukemia. Dasatinib works as a BCR-ABL and ARC-kinase family inhibitor (Lindauer & Hochhaus, 2018). Silodosin is an FDA approved drug used in the treatment of benign prostatic hyperplasia, the drug is an alpha blocker that binds with high selectivity to alpha1A-adrenergic receptors (Rossi & Roumeguere, 2010). SGI-1027 is an experimental drug which is a highly lipophilic quinoline-based DNA methyltransferase 1 enzyme inhibitor with the potential to reactivate tumor suppressor genes. It was seen that SGI-1027 could induce a mild pro-apoptotic effect in human hepatocellular carcinoma cells in vitro. (Sun et al., 2018). Another experimental drug is NSC319726, which inhibits growth in cancer cells expressing mutant P53 (Yu et al., 2012). UNC0631 is an experimental G9a/GLP inhibitor, the G9a-GLP complex mediates dimethylation of histone 3 on lysine 9. It was seen that inhibition of G9a resulted in different epigenetic signatures in cancer cells causing proapoptotic effects (Chen et al., 2019).
The administration of Pomalidomide alone is toxic to both cell lines in a concentration-dependent manner (figure 2). It appears that there is an enhanced effect when combined with hyperthermia, which can be seen from concentrations starting at 0.5 µM. Dasatinib appears to have relatively low toxicity in both cell lines compared to the other drugs tested. When Dasatinib is combined with hyperthermia treatment, an enhanced effect is seen at a concentration of 1 µM in both cell lines. Results show that the addition of Silodosin alone is not toxic to both cell lines. Hyperthermia treatment has an enhanced effect at higher concentrations in both cell lines, however, HeLa cells appear to be more sensitive to the combined effect. SGI-1027 has a high toxicity in both cell lines, HeLa cells appear
5 to be more sensitive than SiHa cells. In combination with hyperthermia treatment, an enhanced effect can be seen from 0.25 µM in HeLa, and 0.5 µM in SiHa cells. NSC319726 has a high toxicity in both Hela and SiHa cells, at starting concentrations of 0.25 µM. It can therefore not be said with high reliability whether hyperthermia strengthens the treatment. The results show that the addition of UNC0631 is not toxic to HeLa cells, however, low levels of toxicity are shown in SiHa cells. With the addition of hyperthermia, an enhanced effect occurs at all concentrations, in a concentration-dependent manner. Based on the results, it can be briefly said that the drugs: Pomalidomide and UNC0631 seem the most promising drugs for the clinic, based on the high cell toxicity in combination with hyperthermia, and the low cell toxicity of the drug alone.
3. Aminophylline, Zalcitabine, Ketoprofen, and Nebivolol make human cervical cancer cells more sensitive to the effects of hyperthermia but stimulates cell division.
Aminophylline, Zalcitabine, Ketoprofen, and Nebivolol are all FDA approved drugs. Aminophylline is an adenosine receptor blocker, phosphodiesterase inhibitors, and a histone deacetylase activator, which is used in the treatment of lung diseases, such as chronic bronchitis, asthma and COPD (Gondal & Zulfiqur, 2019). Zalcitabine is a potent nucleoside analog inhibitor, which inhibits reverse transcriptase in HIV (Devineni & Gallo, 1995). Ketoprofen is a Prostaglandin G/H synthase 1 and 2 inhibitor, which induces anti-inflammatory effects. Ketoprofen is used in the treatment of osteoarthritis and rheumatoid arthritis (Kantor, 1986). Nebivolol is a β1-adrenergic receptor antagonist, which is used in the treatment of hypertension (Fongemie & Felix-Getzik, 2015).
The addition of Aminophylline alone stimulates cell division, in a concentration-dependent manner, however, Aminophylline in combination with hyperthermia is toxic to both cells lines. The administration of Zalcitabine to HeLa does not induce toxic effect, however, is stimulates cell division in SiHa cells. When zalcitabine is used in combination with hyperthermia, cell division is stimulated in both cell lines. Ketoprofen is not toxic in both cell lines. In combination with hyperthermia, cell division is stimulated in HeLa cells at a concentration of 0.5 µM. Administration of Nebivolol is toxic to HeLa cells, however, cell division is stimulated in SiHa cells. The addition of hyperthermia results in cell division in HeLa cells, at a concentration of 0.25 µM, although it causes cell death in SiHa cells at concentrations of 0.25 µM and 0.50 µM.
Based on the data, it can be concluded that Aminophylline, Zalcitabine, Ketoprofen, and Nebivolol are not suitable candidates to improve hyperthermia treatment.
Table 2. Drug selection with target and state of development.
Drugs Targets State of development
SGI-1027 DNA methyltransferase 1 Inhibitor Experimental
NSC319726 P53 Experimental
UNC0631 G9a/GLP inhibitor Experimental
Pomalidomide Angiogenesis Inhibitor FDA approved
Dasatinib BCR-ABL inhibitor and Src family tyrosine kinase inhibitor
FDA approved
Silodosin Alpha1A-adrenergic receptor antagonist FDA approved
Aminophylline Phosphodiesterase inhibitor, adenosine receptor blocker and Histone deacetylase activator
FDA approved
Zalcitabine Reverse transcriptase inhibitor in HIV FDA approved
Ketoprofen Prostaglandin G/H synthase 1/2 inhibitor FDA approved
6 0 5 10 15 20 Silodosin Zalcitabine Aminophylline Dasatinib Nebivolol Ketoprofen Pomalidomide SGI-1027 NSC319726 UNC0631 TER Dr u g
TER of top hits
SiHA HeLa
SiHa
HeLa
SiHa HeLa
SiHa + HeLa
Figure 1. (A) The TER values of all kinase inhibitor drugs analyzed in the drug screen of SiHa and HeLa cells, the broken line shows the DMSO control, a TER higher than 1.4 in SiHa and 1.3 in HeLa indicates a sensitizer. A TER of less than 1.2 in SiHa and 1.1 in HeLa indicates a protector. (B) The TER values of all top hits used in our validation experiment for both SiHa and HeLa. (C) The subdivision of cellular processes/pathways on which the sensitizing drugs interact in SiHa, HeLa, and SiHa + HeLa combined. The broken line shows the percentage of sensitizers in the total screen.
A.
B.
SiHa
HeLa
7 0 0.25 0.50 1 0 0.25 0.50 1 0 20 40 60 80 100 120 140 Pomalidomide normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱✱ ✱✱ ✱✱ ✱✱ 0 0.25 0.50 1 0 0.25 0.50 1 0 20 40 60 80 100 120 140 SGI-1027 normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱✱ ✱✱ ✱✱ ✱✱ 0 0.25 0.50 1 0 0.25 0.50 1 0 20 40 60 80 100 120 140 Aminophylline normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱ ✱✱✱ ✱✱ ✱✱✱ ✱✱✱ ✱✱ 0 0.25 0.50 1 0 0.25 0.50 1 0 20 40 60 80 100 120 140 NSC319726 normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱ 0 0.25 0.50 1 0 0.25 0.50 1 0 20 40 60 80 100 120 140 UNC0631 normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱✱✱ ✱✱✱ ✱✱✱ ✱✱✱ ✱✱✱ ✱✱✱ 0.00 0.25 0.50 1.00 0.00 0.25 0.50 1.00 0 20 40 60 80 100 120 140 Nebivolol normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱✱✱ ✱✱ ✱✱ 0.00 0.25 0.50 1.00 0.00 0.25 0.50 1.00 0 20 40 60 80 100 120 140 Silodosin normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱✱✱ ✱✱✱ ✱✱ 0 0.25 0.50 1 0 0.25 0.50 1 0 20 40 60 80 100 120 140 Zalcitabine normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱ ✱ ✱ 0 0.25 0.50 1 0 0.25 0.50 1 0 20 40 60 80 100 120 140 Dasatinib normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱✱ ✱ 0.00 0.25 0.50 1.00 0.00 0.25 0.50 1.00 0 20 40 60 80 100 120 140 Ketoprofen normalized HT Concentration (μM) N o rm al iz ed s u rv iv al ( % ) HeLa -HT HeLa +HT SiHa -HT SiHa +HT ✱✱✱
Figure 2. The normalized survival of the validated drugs in combination with and without hyperthermia treatment in SiHa and HeLa cell lines, in a concentration range of 0 µM, 0.25 µM, 0.5 µM and 1 µM. * = p <0.05, ** = p <0.01, *** = p <0.001.
8
DISCUSSION
It has never been systematically investigated which kinase inhibitor drugs could potentially improve the effects of hyperthermia treatment. This study, therefore, aimed to systematically evaluate the combinatorial effects of a wide variety of kinase inhibitor drugs and hyperthermia. This study investigated ten potentially effective kinase inhibitor drugs, which have emerged from our drug screen of more than 1400 kinase inhibitor drugs. The data of the drug screen showed that kinase inhibitor drugs can have a sensitizing or protective effect on hyperthermia treatment in human cervical cancer, and that the sensitizing effect of the kinase inhibitor drugs on hyperthermia treatment is not related to one specific cellular process or pathway. A selection of the most promising drugs was made, based on the TER and whether the drug is FDA approved, these drugs were then validated in multiple concentrations.
Our drug validation experiment showed that almost all drugs enhance the effect of hyperthermia to different degrees (table 3). Our data suggest that UNC0631 is the most promising experimental drug, and Pomalidomide is the most promising FDA approved drug for further research. These drugs both show a favorable toxicity profile, in both cell lines. UNC0631 is already toxic in low concentrations when combined with hyperthermia, furthermore, the drug without hyperthermia treatment shows almost no cell toxicity at low concentrations. Pomalidomide is more toxic at equal concentrations compared to UNC0631, however, this FDA approved drug does show an enhanced effect at concentrations from 0.5 µM in both cell lines. The drugs SGI-1027, NSC319726, Silodosin, Dasatinib, Aminophylline, Zalcitabine, Ketoprofen, and Nebivolol are not of interest for the follow-up study. SGI-1027, NSC319726, Silodosin, and Dasatinib show a favorable toxicity profile, but this is only reached at high concentrations and is cell line specific. The drugs Aminophylline, Zalcitabine, Ketoprofen and Nebivolol showed an enhanced effect in combination with hyperthermia, however, these drugs did stimulate cell division to different degrees. More research should therefore be conducted, as stimulating cell division in cancer therapy is not a beneficial side effect.
Discrepancies occurred between the drug screen and the drug validation experiments, regarding the TER values. This can be explained by the fact that all drugs were incubated in the drug screen for 72 hours, whereas this study maintained other incubation times relevant to the clinic. Another limitation in this study was that all drugs were administered 30 minutes before the hyperthermia treatment, however some drugs act on gene expression, which would require them to be administered at least 24 hours in advance to achieve an effect of the drug. Another important note is that triplicates have been used for statistical analysis, and therefore results should be interpreted with caution.
For follow-up research, a trade-off should be made between a high-risk high-reward approach or a low-risk low-reward approach. UNC0631 shows the most favorable toxicity profile, however, G9a/GLP inhibitors have only just made their entry into in vivo research, the path to clinical trials may take decades (Ma et al., 2020). Pomalidomide, however, is already an FDA approved drug with less of an effect than UNC0631. This drug can therefore quickly result in a new combinatorial treatment. More research, however, is still needed to achieve this. UNC0631 and Pomalidomide should first be tested in multiple cell lines to determine if the combinatorial effect is also mediated in other cancer types. A logical next step would be to investigate similar drugs, with the same targets, to see if they produce the same combinatorial effect. Then omics experiments should be performed to determine the mechanistic effect of the drug, to investigate which cellular component or pathway is responsible for the thermosensitizing effect.
9 The thermosensitizing effect is expected to be polygenic, since the cells have already developed an extensive protection mechanism against heat stress, which is expected to be redundant. Thus, in order to induce an actual thermosensitizing effect, one would have to switch off different combinations of genes, which can be achieved by gene and histone methylation. Both drugs should furthermore be examined whether the combinatorial effect is also enhanced in the presence of radio- and chemotherapy, since the hyperthermia treatments cannot replace the conventional methods yet. In summary, this study has systematically looked at how hyperthermia treatment can be improved using kinase inhibitors. One of the most promising drugs found were UNC0631 and Pomalidomide.
Table 3, TER Values in SiHa and HeLa of preliminary study and present study. The TER is indicated for the concentration at which it was highest in the present study.
TER Derived from
drug screen
TER
Derived from drug validation experiments
Potential for follow-up research
SiHa HeLa SiHa HeLa
UNC0631 16.3 1.6 3.7 (0.5 µM) 3.2 (1.0 µM) Yes NSC319726 3.2 3.2 2.5 (0.5 µM) 2.5 (0.5 µM) No SGI-1027 2.0 1.7 8.5 (1.0 µM) 5.3 (0.5 µM) No Pomalidomide 1.8 1.4 1.3 (1.0 µM) 1.2 (1.0 µM) Yes Ketoprofen 1.6 1.5 1.0 (0.25 µM) 1.0 (0.25 µM) No Nebivolol 1.7 1.4 1.2 (0.5 µM) 1.1 (1.0 µM) No Dasatinib 1.6 1.6 1.2 (1.0 µM) 1.2 (1.0 µM) No Aminophylline 1.6 1.5 1.3 (0.5 µM) 1.3 (0.5 µM) No Zalcitabine 1.5 1.5 1.1 (0.25 µM) 0.9 (0.5 µM) No Silodosin 1.5 1.5 1.1 (0.5 µM) 1.4 (0.5 µM) No
AKNOWLEDGMENTS
I would like to extend my sincere thanks to Enzo Scutigliani, who guided me daily in the lab and while writing this thesis. Through his excellent guidance and constructive criticism, I notice that I have been able to make enormous steps in scientific writing. I’d also like to extend my gratitude to Przemek Krawczyk, the head of the research group, for his insightful suggestions and supervision. I also had great pleasure of working with Haibin Qian, who continued some of the experiments during the coronavirus pandemic.
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