La aceleración de los ciclos de la moda ligada a la globalización

For this thesis, several neuroblastoma cell lines were treated in vitro and in vivo with BET inhibitors. Thereby a large number of different experimental approaches were illuminating different biological aspects of BET inhibition in neuroblastomas. One central question of this work was to estimate how neuroblastoma cells with certain genetic features will respond to BET inhibitor treatment, aiming to be able to suggest a potential therapy with a BET inhibitor to the suitable patients based on the biology of their tumor. For comparison of in vitro and in

vivo results the focus will be on the cell lines used in the animal experiments (IMR5/75, LS,

NBL-S, SH-SY5Y) as well as NGP as example for a cell line not responding to BET inhibition. Viability analysis of the neuroblastoma cell lines upon treatment with the BET inhibitors JQ1 and BAY1238097 revealed a higher effectivity of the inhibitor JQ1 for all cell lines. The cell lines LS and IMR5/75 were both classified as sensitive upon JQ1 treatment with similar EC50- values for JQ1 and BAY1238097. NGP cells presented as highly resistant with both inhibitors.

147 NBL-S cells were classified as intermediate responders to JQ1 treatment and an intermediate- resistant response upon BAY1238097 therapy. Despite the lower effectivity of BAY1238097, this inhibitor was chosen for the in vivo experiments due to its superior pharmacokinetic properties, whereas in vitro experiments were performed with JQ1.

Further insight into the functions of BET inhibition was gained by FACS-analyses used to estimate cell cycle and cell death of JQ1-treated neuroblastoma cells. An arrest in the G1 phase of the cell cycle was shown for IMR5/75, LS and NBL-S, which was strongest in IMR5/75 cells. This effect can be explained with the high proliferation rate of the IMR5/75 cells. IMR5/75 cells have a doubling time lower than 24h, in this case the cells are accumulating faster in G1 after completing the previous cell cycle than cells with a longer doubling time. An increase in cell death was observed in few MYCN amplified JQ1-treated cell lines only, most prominent in IMR5/75 cells. Despite the high levels of cell death, IMR5/75 were regrowing fast after washout of JQ1, showing that the long-term negative effects on cell proliferation of a shRNA induced

MYCN knockdown were stronger than JQ1 treatment. These observations indicate that the

effects induced by JQ1 treatment are temporary, especially for fast dividing IMR5/75 cells. In two independent publications a direct regulation of MYCN by BRD4 in neuroblastoma was stated [121, 122], suggesting MYCN as a target gene of BET inhibition. To evaluate this, the MYCN protein expression was tested by two methods in a larger panel of MYCN amplified neuroblastoma cell lines, showing a reduction of MYCN in most of the cell lines. Especially for IMR5/75 and LS, the effects on the MYCN protein levels were minimal or ambiguous. For both cell lines, MYCN is also not listed among the differentially expressed genes identified by RNA- sequencing. For IMR5/75 and SK-N-BE(2)c treated with BRD4 siRNA the MYCN expression was not affected, leading to the conclusion that BRD4 is not absolutely required for MYCN expression in neuroblastoma.

Based on the data collected as part of this thesis it was not possible to finally clarify which factors are pivotal for sensitivity to BET inhibition. Though, some relations have been observed between the cell lines and their response to BET inhibitor treatment. One observation is that all three MYCN amplified cell lines with a doubling time of 24h and below (IMR5/75, SK-B- BE(2)c, IMR32) are BET inhibitor sensitive and show a prominent G1-arrest and high levels of cell death (Figure 67, left side). It is supposed that the fast cell cycle turnover causes a fast and strong accumulation of the cells in G1, presumably by the reduction of the transcription of cell cycle promoting genes, and an increase of cell death earlier than for cell lines dividing more slowly. On the other hand, the high proliferation rate goes in hand with a fast regrowth after the end of the therapy. Hence, this group of sensitive cells is characterized by early growth inhibitory effects that can be evaded immediately after removing the inhibitor and are thereby no permanent effects. For long term effects it is supposed that additional genetic aberrations

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besides MYCN amplification are priming cells for BET inhibitor sensitivity (Figure 67, right side). This is the case for LS cells with amplifications of the cell cycle regulating genes CDK4,

MDM2 and CCND1. Amplifications of CDK4 and MDM2 are also detectable in BET inhibitor

sensitive TR14 and in resistant NGP cells. In detail, a comparison of different cell lines for the definition of general rules is difficult as not enough neuroblastoma cell lines with comparable patterns of genetic aberrations could be analyzed.

For clinical settings, it is not only necessary to identify the most sensitive tumors, but also to define the group of tumors that will be highly resistant to avoid needless therapies. Among the group of MYCN amplified and JQ1 resistant cell lines are NGP and KELLY, both have an activation of TERT (Figure 67, bottom part). Besides the TERT activation, which was present solely in those two of the MYCN amplified cell lines, the genetic background of KELLY and NGP is quite different. In NGP, as described above, the cell cycle promoting genes CDK4 and

MDM2 are amplified. KELLY harbor an inactivating mutation in the TP53 gene and an

activating mutation in the ALK gene. A TERT activation and an ALT phenotype are two ways how neuroblastoma cells enable a stabilization of telomere length and avoid cellular senescence [164]. Among the MYCN non-amplified neuroblastoma cell lines, two further cell lines with telomere stabilization and resistant to JQ1 treatment were identified: GIMEN cells have a TERT activation and SK-N-FI cells have an ALT phenotype. Those results hint that the presence of long telomeres might be involved in BET inhibitor resistance, independent from the MYCN-amplification status. However, a TERT activation or ALT phenotype are not consistent in predicting the resistance upon BET inhibitor therapy, as there are cell lines harboring those genetic aberrations that are JQ1 sensitive or intermediate responding, such as SK-N-AS with an activation of TERT or CHLA-90 with an ALT phenotype. It is supposed that the resistant cell lines harbor further genetic aberrations enabling those cells to grow upon BET inhibitor therapy. Also there are several MYCN amplified neuroblastoma cell lines resistant upon JQ1 treatment without TERT activation or an ALT phenotype

Altogether, clear definitions based on the genetic background of the cell lines could be formulated neither for BET inhibitor resistance nor for the identification of highly responsive cell lines. The panel of 23 neuroblastoma cell lines with diverging genetics was heterogeneous and too small to analyze universally valid hypotheses regarding BET inhibitor sensitivity statistically.

149 Figure 67: Resistance and sensitivity of neuroblastoma cells to BET inhibition. Genetic factors and growth characteristics supposed to be relevant factors for sensitivity or resistance of neuroblastoma cells upon JQ1 treatment. The categories sensitive, intermediate and resistant are based on the results of the viability screening (section 5.2.1). DT= doubling time; amp= amplification; NA= non-amplified. Crossed out cell lines show contrary behavior upon BET inhibition that other cells with similar genetic background.

In vivo, mice with LS deriving xenograft tumors showed a seven days prolonged survival upon

treatment with BAY1238097, which is highly effective related to a total experimental duration of up to 30 days. No tumor growth inhibiting effects were detectable for tumors deriving from IMR5/75 cells although those cells had been highly sensitive to BET inhibitor treatment in vitro. The difference between both cell lines can be explained as those fulfill different criteria for JQ1 sensitivity (Figure 67), leading to a long-term effect of BET inhibition on LS cells and non- permanent effects on IMR5/75.

The experiments with IMR5/75 xenograft tumors were performed in two different mouse strains, however the validity of the results was limited due to variability of tumor onset and inhomogeneity of tumor growth especially among the control group. Besides the explanation for non-permanent growth inhibitiong effects in BET inhibitor-treated IMR5/75 cells and tumors, it cannot be excluded that a potential inhibitory effect for tumor growth was obscured by the inconsistent control group.

Summarizing the results for BET inhibitor treatment in vitro and in vivo, at present it is not possible to draw conclusions from a BET inhibitor sensitive cell culture setting to a successful therapy in vivo.

SK-N-AS

CHLA-90

NGP

Kelly

GIMEN

SK-N-FI

resistant

TERT activation or ALT phenotype

Type 2 sensitive

MYCN amp, CDK4/MDM2 amp

LS

TR14

NGP

IMR5/75

IMR32

SK-B-BE(2)c

Type 1 sensitive

sensitive

MYCN NA, TERT

intermediate

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