1 Annemijn Cassée The Effect of Hyperthermia in Tumors with IDH1MT
Universiteit van Amsterdam Annemijn Cassée - 10649794
[
THE EFFECT OF
HYPERTHERMIA IN TUMORS
WITH ISOCITRATE
DEHYDROGENASE 1
MUTATION
]
Various cancer types are associated with the IDH1 mutation. Under normal conditions, IDH1 wilde type catalyzes isocitrate into a-ketoglutarate in presence of NADP+. In contrast, IDH1 MT catalyzes a-ketogluarate into D-2hydoxyglutarate (D-2HG) in presence of NADPH, which eventually induces malignant transformations. However, IDH1 mutation tends to be more sensitive for treatment since NADPH normally detoxificates the cell and scavenges the oxigen levels. Mutated IDH1 cells have an insufficienty of NADPH. Moreover, the homologous recombination in the IDH1 mutation is inhibited, which also makes the cell more sensitive for treatment. Hyperthermia is a therapy in which tumor cells are heated between 41-42.5°C. This causes an additional effect in combination with radiation. In this research, colony-forming assays are performed to investigate the effect of hyperthermia (in combination with ionizing radiation) on the IDH1 mutation. The results show that hyperthermia inhibits the IDH1 mutation and the IDH1 wildtype. Furthermore, it enlarges the effect of IR. The IDH1 mutant was more affected by the treatment than IDH1WT. More clinical research is necessary to determine if hyperthermia is effective to inhibit IDH1 MT in vivo. Further research should investigate whether hyperthermia inhibits the IDH1 MT because it further inhibits HR or because it activates the NADPH consumption.Various cancer types are associated with the IDH1 mutation. Under normal conditions, IDH1 wild type (IDH1 WT) catalyzes isocitrate into a-ketoglutarate in presence of NADP+. In contrast, IDH1 MT catalyzes a-ketogluarate into D-2hydoxyglutarate (D-2HG) in presence of NADPH, which eventually induces malignant transformations. However, IDH1 mutation tends to be more sensitive for treatment since NADPH normally detoxificates the cell and scavenges the oxigen levels. In mutated IDH1 cells (IDH1 MT), there is insuffient NADPH to detoxificate the cell well enough. Moreover, the homologous recombination in the IDH1 mutation is inhibited, which also makes the cell more sensitive for treatment. Hyperthermia is a therapy in which tumor cells are heated between 41-42.5°C. This causes an additional effect in combination with ionizing radiation (IR). In this research, colony-forming assays are performed to investigate the effect of hyperthermia (in combination with ionizing radiation) on the IDH1 mutation. The results show that hyperthermia inhibits the IDH1 mutation and the IDH1 WT. Furthermore, it enlarges the effect of IR. The IDH1 MT was more affected by the treatment than IDH1 WT. More clinical research is necessary to determine if hyperthermia is effective to inhibit IDH1 MT in vivo. Further research should investigate whether hyperthermia inhibits the IDH1 MT due to the inhibition of HR
Introduction
Mutations in the isocitrate dehydrogenase 1 and 2 (IDH1MT and IDH2MT) occur in various types of cancer including glioma, acute myeloid leukemia (AML) cholangiocarcinoma, chondrosarcoma and others [ CITATION Mol15 \l 1043 \m Mol151] (see table 1 for an overview of mutation frequency). IDH1 and IDH2 are homodimeric enzymes that reversibly catalyze isocitrate into a-ketoglutarate (a-KG) and CO2. NADP+ serves as a co-factor to mediate this reaction. [ CITATION Yen10 \l 1043 ]. IDH1 and IDH2 share significant structural similarity but have differences in the subcellular localization. IDH1 is localized in both peroxisomes and cytosol, whereas IDH2 is active in the mitochondria [ CITATION Yen10 \l 1043 ]. When mutated, the active sites of the enzymes changes by altering a single amino acid (R100 and R132 in IDH1 and R140 and R172 in IDH2) (Molenaar, et al., 2014). This leads to a gain of function in the IDH1/2MT. Instead of isocitrate, IDH1/2MT convert a-KG into D-2-hydroxyglutarate (D-2HG) [ CITATION Mol151 \l 1043 ]. Furthermore, instead of NADP+, NADPH serves as a co-factor and is converted into NADP+. D-2HG accumulates in the cancer cells and competitively inhibits or activates a-KG-dependent dioxygenases, which are involved in many cellular processes. This inhibition or activation tends to be responsible for global DNA hypermethylation, which alters gene expression and eventually induces malignant transformation [ CITATION Mol15 \l 1043 \m Mol151].
Interestingly, glioma that carry the IDH1/2MT are associated with a prolonged survival rate of
the patients, as compared to the IDH1/2WT (Molenaar, et al.,; 2014; Houillier, et al., 2010).
Research suggests that tumor cells containing the IDH1/2MT tends to be more sensitive to
ROS-levels induced by radiation (IR) and chemotherapy, in comparison to tumor cells with IDH1/2WT (Tran, et al., 2014; Okita, et al., 2012; Molenaar, et al., 2014; Houillier, et al., 2010).
According to those studies, these sensitivity to therapy may cause the prolonged survival rate. As previously stated, IDH1//2MT consumes NADPH instead of production. Research
shows that the production of NADPH in IDH1MTdecreases is decreased by 50% [ CITATION Ata11 \l 1043 \m Ble10]. NADPH Nnormally, NADPH detoxificates the cell and scavenges the oxigen levels through the production of glutathione (GSH) [ CITATION Hou10 \l 1043 \m Koe03]. Diminishing the NADPH levels and thus the reduction of GSH leads to more oxidative stress that can ultimately causes DNA damage and apoptosis [ CITATION Hou10 \l 1043 ]. In chemotherapy and radiation, the oxidative stress is generally increased as a result of accumulation of ROS. In these periods of extreme stress, the NADPH demand may exceeds the NADPH production and thereby, the detoxifying capacity of the cancer cell is insufficient when demand is at its peak (such as after a sufficient IR dose) (Molenaar, et al., 2014; Houillier, et al., 2010). Therefore, IDH1/2MT are more sensitive for chemotherapy and
radiation compared to IDH1WT [ CITATION Mol15 \l 1043 ].
Figure 1 the molecular effects of IDH1/2MT in the cell (Molenaar, et al., 2014). IDH1/2MT induce malignant
transformation through the accumulation of D-2HG in the cell that induces DNA hypermethylation. However IDH1/2MT have an increased sensitivity for treatment since the NADPH production is decreased.
An additional treatment to sensitize cancer cells for radiation and chemotherapy is hyperthermia [ CITATION Oei17 \l 1043 ]. Hyperthermia is a therapy in by which the cancer cells are heated above the normal physiological body temperature. Hyperthermia is mostly used for approximately 1 hour at 41-42.5°C., which Such therapy is named mild-hyperthermia [ CITATION Oei17 \l 1043 ]. Mild mild-hyperthermia alone as a single modality has been suggested not to be an effective therapy for cancer [ CITATION Hil02 \l 1043 \m Wus02]. However, there has been observed an additional interaction between heat and radiation dose as well as heat and various cytostatic treatments [ CITATION Wus02 \l 1043 ]. A body of extensive studies have showed that hyperthermia may interfere with DNA metabolism and that hyperthermia in combination of radiation or chemotherapy leads to DNA damage (Oei, et al., 2015). This mightay suggest that hyperthermia influences the DNA repair mechanisms. Indeed, studies showed that hyperthermia interferes with homologous recombination by temporaryaly downregulating the BRCA2 protein [ CITATION Oei17 \l 1043 \m Kra11]. Homologous recombination is a Doube Strand Break (DSB) repair mechanism in mammalian cells, which requires a homology template. Therefore, homologous recombination is only active only in the S- and G2-phases (Oei, et al., 2015). The BRCA2 protein interacts with other major proteins that are involved in the homologous recombination [ CITATION Oei17 \l 1043 \m Pow03]. This is in particular the RAD51 protein, which is critical in the initiating the first reaction of homologous recombination. RAD51 protein is only active in association with the BRCA2 protein. Thus, the degradation of BRCA2 protein in hyperthermia induces a reduction in homologous recombination. Since radiation induces DSB’s which normally can be repaired by homologous recombination, hyperthermia increases the sensitivity to radiation [ CITATION Pow03 \l 1043 ].
Interestingly,Until recently, recent research showed shows that the accumulation of D-2HG in IDH1MT induces a defect in the homologous recombination repair of cancer cells
(Sulkowski, et al., 2017). Since the accumulation of D-2HG in IDH1MT as well as hyperthermia
inhibits the DBS repair mechanism of homologous recombination, we hypothesizeed that hyperthermia might serve as an effective treatment in cancer cells with an IDH1MT.
No research has been performed to examine the effect of hyperthermia on the IDH1 MT
cancer cells. Thus, the aim of this study is to examine whether IDHWT and IDH1MT are
sensitive for treatment with hyperthermia (in combination with ionizing radiation) and whether IDH1MT are more sensitive to hyperthermia compared to IDH1WT.
Material and Methods
For this research, we used the HCT116 IDH1WT/MT and HCT116 IDH1WT/WT knock-in cells to
perform a colony-forming assay. Both of these cells were cultured in McCoy’s 5A medium (Gibco; Life Technologies; Thermo Fisher Scientific) in 5% CO2 at 37°C. The medium was
supplemented with 10% FBS and 100 units/mL penicillin. Depending on the dose of radiation (IR), the cells were seeded between 200-2000 cells/cm2. Higher concentrations were
necessary when higher irradiation radiation doses were performed to obtain sufficient amount of colonies. The cells were irradiated with a 137Cs source (Department of
Experimental Oncology and Radiobiology, Academic Medical Center, University of Amsterdam) 24 hours after plating. The radiationirradiation doses varied between 2 to 6 Gy. An hour after irradiationIR, some cells were heated at 42°C for 1 hour. Cells were fixed and stained at 7-10 days after plating with a mixture of 0.05% crystal violet (Merck) and 6% glutaraldehyde (Merck) for 24 hours at room temperature. The cells were manually counted using a stereoscope (Leica MZ6, Leica Microsystems; ref. 33). The data are expressed as the clonogenic fraction, which is the amount of colonies that where counted, divided by the amount of cells plated. Control experiments where performed with no IRirradiation doses and no heating above physical body temperature (37°C).
Statistical analysis
Data of the experiments were processed in Excel and analyzed using GraphPad Prism 6. P values were obtained using a non-parametric t-test in Graphpad Prism 6 on the difference of treated cells in combination with hyperthermia between IDH1WT and IDH1MT. The same
statistical analysis was used to obtain the P value of the difference between heated and non-heated cells at different radiation doses of the IDH1WT and IDH1MT. Data shown are
representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Results
Hyperthermia inhibits the IDHWT and IDH1MT in vitro.
To investigate the effect of hyperthermia (HT) alone, we performed colony-forming assays in the IDH1WT and IDH1MT cells. The cells treated with HT (42°C) have a significantly lower
survival compared to non-treated cells (37°C) (see Figure 2A). These data show that hyperthermia alone may affect the cell in vitro. Thus, these results suggest that HT sensitizes the cells to inhibit IDHWT and IDH1MT in vitro. Because hyperthermia in combination with IR
already has been tested and has shown to be effective, we next hypothesized that the combination of hyperthermia and IR could be more effective.
Hyperthermia increases the effect of ionizing radiation ( IR) on IDH1WT and IDH1MT.
To investigate the effect of HT after IR, we performed colony-forming assays in the IDH1WT
and IDH1MT cells. The cells treated with HT in combination with IR have a significantly lower
survival rate compared to non-heated cells. This effect was illustrated after treatment with 2 and 4 Grey of radiation (see Figure 32B and 2C). Irradiation IR with 6 and 8 Grey were was not included in the results since the none of the cells survived. Taken together, these results confirm that HT increases the effect of IR in the IDHWT and IDH1MT in vitro. Since we showed
that IDH1WT and IDH1MT were affected in differently, we next examined whether IDH1MT was more sensitive to hyperthermia compared to IDH1WT.
IDH1MT cells are more sensitive to hyperthermia (in combination with IR) than IDH1WT.
To investigate whether IDH1MT cells are more sensitive to hyperthermia, we repeated
colony-forming assays in IDH1WT and IDH1MT. A significantly lower survival was observed in the IDH1 MT cells when treated with hyperthermia. Furthermore, a significant lower survival of the
IDH1MT was also observed with the combination treatment of hyperthermia and irradiation
radiation (Fig. 43). These results confirm that IDH1MTare is more sensitive to hyperthermia
7 Figure 3
A) Hyperthermia sensitizes the IDH1WT cells to 2 and 4 Grey of IR. The figure shows a colony-forming assay after 2 and 4 Grey with or without hyperthermia in the IDH1 WT cells. A significantly lower clonogenic fraction is measured after heated for 1 hour at 42°C. P values were obtained using a non-parametric t-test on the difference between heated and non-heated cells for IDH1WT cells. P-value **** for the difference after 2 Grey. P-value *** for the difference after 4 Grey of IR. Furthermore, when treated with more IR (4 Grey compared with 2 Grey), the IDH1WT cells are more inhibited.
B) Hyperthermia sensitizes the IDH1MT cells to 2 and 4 Grey of IR. The figure shows a colony-forming assay after 2 and 4 Grey with or without hyperthermia in the IDH1 M T cells. A significantly lower clonogenic fraction is measured after heated for 1 hour at 42°C. P values were obtained using a non-parametric t-test on the difference between heated and non-heated cells for IDH1WT cells. P-value **** for the difference after 2 Grey. P-value *** for the difference after 4 Grey. Furthermore, when treated with more IR (4 Grey compared with 2 Grey), the IDH1MT cells are more inhibited.
Figure 2
Hyperthermia inhibits the clonogenic fraction of the IDH1WT
and IDH1MT
in vitro after no radiation dosesirradiation dose. The figure shows a colony-forming assay after 0 Grey with or without hyperthermia of IDH1WT and IDH1MT3H. A significantly lower clonogenic fraction is measured after heated for 1 hour at 42°C. P values were obtained using a non-parametric t-test on the difference between heated and non-heated cells for IDH1WT and IDH1MT. P-value ** for both IDH1WT and IDH1MT).
Figure 43
A) IDH1MT are more affected than IDH1WT when treated with irradiation radiation doses higher than 2 Grey. The figure shows a colony-forming assay of IDH1WT and IDH1MT treated with different doses of radiation. This difference is most measured at 2 Ggrey (p-value ***). P values were obtained using a non-parametric t-test between IDH1MT and IDH1WT after treatment with hypertermia and different doses of radiationirradiation.
B) IDH1MT are more affected than IDH1WT affected when treated with hyperthermia after different irradiation radiation doses (2-4 Grey) than IDH1WT. The figure shows a colony-forming assay of IDH1WT and IDH1MT cells after heated for 1 hour at 42◦C and after 0-6 Grey. of IDH1WT and IDH1MT. A significantly lower cClonogenic fraction is observed at different doses of irradiation IR of the IDH1MT cells. This difference is most observed at 2 Grey. A significant lower survival rate of IDH1MT is measured after treatment with hypertermia and no irradiatonIR. P values were obtained using a non-parametric t-test between IDH1MT and IDH1WT after treatment with hypertermia and different doses of irradiation.
Discussion
The This present study is the first to investigate the effect of hyperthermia on the IDH1WT
and IDH1MT cells. In this study, we provided evidence that hyperthermia inhibits the IDH1MT
and IDH1WT cells. Furthermore, we tested the effect of hyperthermia in combination with IR.
We provided evidence that hyperthermia in combination with IR, is an effective treatment to inhibit IDH1MT and IDH1WT. More interestingly, this research showed that IDH1MT cells are
more sensitive to the combination of hyperthermia and IR compared to IDH1WT cells. More
research is necessary to determine if his effect remains when the effect of hyperthermia on the IDH1MT is tested in vivo.
The resultsThis study showed that hyperthermia affects the IDH1MT and IDH1WTcells in vitro.
This effect is also measured without any radiation doses. This suggests that hyperthermia induces DNA damage, not solely due to the inhibition of homologous recombination. This is in contrast of the study of Oei., et al (2017) which states that hyperthermia does not induce Ddouble Sstrand Bbreaks (DSB’s) and does not induce DNA damage directly. However, previous research stated that heat (> 41.5°C) triggers focal phosphorylation of histone γH2AX (histone H2AX phosphorylated at serine 139) which are similar to the so-called ionizing radiation induced foci (IRIF) [ CITATION Tak04 \l 1043 ]. Normally, γH2AX measurement is used as a reliable and sensitive method to measure double strands breaksDSB’s, since the number of γH2AX correlates with the number of DSB’s (Sedelnikova, et al., 2002). The γH2AX correlated positively with the survival rate of different cell lines, indicating that the heat-induced DSBs lead to cell death [ CITATION Tak04 \l 1043 ]. Furthermore, observations have shown that ATM activation occurs after heat-treatment, which is a known reaction in the presence of DSB’s (Takahashi, et al., 2007). However, some investigators reserachers believe the hypothesis that heat induces DSBs are incorrect. This is based on the fact that several proteins (e.g. NBS1, MRE11, RAD50) are directly associated with IR-induced DSB’s and are not been found in foci after heat treatment (Takahashi, et al., 2010). An alternative explanation for the heat-induced γH2AX foci formation is that these formations are a result of chromatin remodeling. However, this possibility has not been confirmed and studies that include experimental chromatin-remodeling treatments detect no γH2AX foci formation [ CITATION Bak03 \l 1043 ]. A possible explanation might be that heat induced DSBs differ from radiation induced DSBs (Takahashi, et al., 2010). Future work is necessary to investigate the mechanism of hyperthermia.
Furthermore, we provided evidence that hyperthermia in combination with IR, is an effective treatment to inhibit IDH1MT and IDH1WT. This study showed that IDH1MT is more
affected by hyperthermia in combination with IR compared to IDH1WT. This affect can be
attributed to the insufficiency of NADPH production in the IDHMT, which makes the cell more
sensitive to treatment (Molenaar, et al., 2014). Research shows that IR induces ROS production, which creates oxidative stress (Molenaar, et al., 2014). Normally, these ROS
levels are scavenged by the activation of NADPH. A recent study provides evidence that HT activates the NADPH oxidase, which converts NADPH into NADP+ [ CITATION Moo10 \l 1043 ]. The effectiveness effect of hyperthermia in combination with ionizing radiation on the IDH1MT cells as well as the IDH1WT cells might be attributed to this activation of the
NADPH oxidase by hyperthermia. Because the IDH1MT already inhibits the NADPH
production, treatment with hyperthermia might cause an additional effect which further inhibits the detoxification of the cell. This might explain why IDH1MT is mostly inhibited by
hyperthermia in combination with ionizing radiation.
However, recent research showed that de accumulation of D-2HG in IDH1MT induces a
homologous recombination defect which might be another explanation for the increased sensitivity of hyperthermia in IDH1MT (Sulkowski, et al., 2017). This might be a possible
explanation since hyperthermia also inhibits the pathway of homologous recombination (Oei, et al., 2015). The application of hyperthermia in the treatment for IDH1MT-depending
cancer types might be most affected due to the complex inhibition of homologous recombination. Further research is required to confirm that the effectivity of hyperthermia on IDH1MT is due to the inhibition on HR or due to the insufficiency in the NADPH production.
Future research should include FK866, an inhibitor of nicotinamide phosphoribosyl transferase that depletes NAD+ [ CITATION JuH16 \l 1043 ]. FK866 in combination with IDH1WT might serve as a positive control to investigate the exact role of the decreased
NADPH production in IDH1MT in the sensitivity to hyperthermia. Furthermore, additional
studies will be needed to investigate whether the effect of hyperthermia sustain when the IDH1 mutant inhibitor AGI-519 is combined with IDH1MT. In combination with simultaneous
treatment of R-2HG, which provide increased 2HG independent of the inhibited mutant enzyme, the role of HR on the sensitivity of hyperthermia can be investigated (Sulkowski, et al., 2017). If the effectivity of hyperthermia in combination with ionizing radiation sustains, this might mean that this effect is due to the homologous recombination defect. In that case, future research should be undertaken to investigate the effect of ionizing radiation in combination with hyperthermia and the PARP-inhibitors BMN-673, olaparib, MK-4827, or rucaparib. These PARP-inhibitors further inhibits homologous recombination and might lead to total lethality of IDH1MT (Sulkowski, et al., 2017).
Because hyperthermia in combination with IR inhibits the IDH1MT, further research should be
alone. Moreover, weI studied wheter the IDH1MT cell is more sensitive to hyperthermia than
IDH1WT cell. This study not only provides evidence for both statements, it also found an
effect of hyperthermia as single modality on the IDH enzyme. This research extends our knowledge of the treatment in various cancer types which includes IDH1132H. Furthermore,
ionizing radiation in combination with hyperthermia showed to be effectiveness effective in IDH1WT cells, which is promising for cancer treatment, since IDH1WTcells are more frequently
present in various other types of cancer.
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