Myc inhibition increases survival of GFAP-V12Ha-Ras mice

To determine the therapeutic impact of systemic Myc inhibition in both progression and maintenance of glioma, we combined our switchable TRE-Omomyc;CMVrtTA mouse with the well-characterized GFAP-V12Ha-Ras (ref. 19), a spontaneous mouse model of multifocal invasive astrocytoma in which the activated form of Ha-Ras is driven by the glial fibrillary acidic protein promoter (GFAP; Fig. 1a). The molecular and pathological progression of disease in GFAP-V12Ha-Ras mice resembles that of diffuse astrocytomas in humans, exhibiting reproducible kinetics of tumour progression, from astroglial hyperplasia (starting from 1–3 weeks of age) through to low- and high-grade gliomas20.

Figure 1: Myc inhibition confers a survival advantage in GFAP-V12Ha-Ras mice. (a) To obtain a mouse model of spontaneous glioma in which Omomyc expression could be activated upon doxycycline treatment, TRE-Omomyc;CMVrtTA mice were crossed with GFAP-V12Ha-Ras mice, giving the triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras. (b) Kaplan–Meier curve showing that Omomyc expression confers a significant survival advantage to V12Ha-Ras expressing mice. Eight-week-old mice, untreated or treated with Omomyc, were monitored for symptom-free survival up to 57 weeks of age.+Omomyc: TRE-Omomyc;CMVrtTA; GFAP-V12Ha-Ras triple transgenics plus doxycycline (n=8).−Omomyc: six vehicle-treated TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras triple transgenics and two CMVrtTA;GFAP-V12Ha-Ras treated with doxycycline, to control for any doxycycline effect (n=8) P=0.014 by log-rank test. No effect of doxycycline was observed on tumour latency. (c) Representative pictures of GFAP immunostaining from the brains of mice included in the survival curve above. The panels on the right are higher magnification images of regions indicated by the black boxes. Control mice present dense GFAP-positive cells compared with Omomyc-treated mice. Full size image

Omomyc was induced in TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras mice from postnatal week 8 with doxycycline and the animals then monitored to determine symptom-free survival. Strikingly, at 57 weeks of age, all Omomyc-expressing mice (8/8) were asymptomatic with no evidence of disease progression (Fig. 1b,c). By contrast, at the same time point only 25% of the untreated control GFAP-V12Ha-Ras mice were still alive, the majority having been euthanized by this time point due to the appearance of progressive ataxia and neurological symptoms, associated with astrocytic hyperplasia and increased intracranial pressure (Fig. 1c).

Of note, mice subjected continuously to Myc inhibition showed no sign of any distress or discomfort, confirming the previously reported well-tolerated and mild side effects elicited by Myc inhibition in normal tissue maintenance and homeostasis16.

Myc inhibition limits growth of transformed neuroprogenitors

To understand how Myc inhibition prevents glioma formation, we first investigated the cellular compartment previously identified in different glioma models as the putative cell of origin of the tumour: the neuroprogenitor (NPG) cells21,22. Recent reports demonstrate that glioma cells share features with neural stem and progenitor cells, including a high proliferation rate, and self-renewal and migration abilities3,23,24. As Myc has a demonstrated role in normal and neoplastic stem cell biology, we assessed the impact of Omomyc expression in primary NPG cells derived from our TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras mice.

Neural progenitor cells were isolated from the subventricular zones (SVZs) of either adult triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras or control double transgenic TRE-Omomyc;CMVrtTA brains and grown as neurospheres in serum-free medium supplemented with growth factors (EGF and bFGF). As expected, the cells stayed strongly positive for GFAP expression (Fig. 2a). In these cultured NPGs, Omomyc mRNA expression was clearly detectable by 48 h of starting doxycycline treatment, whereas no Omomyc signal was detectable in untreated cells (Supplementary Fig. 1). Of note, even though NPG cultures derived from the triple transgenic TRE-Omomyc; CMVrtTA;GFAP-V12Ha-Ras mouse expand significantly more than non-V12Ha-Ras-expressing TRE-Omomyc;CMVrtTA cells, Omomyc expression in Ras-transformed cells profoundly decreased total cell number at 3 days (Fig. 2b). At this same 3-day time point, the control cell culture had barely expanded and Omomyc expression had negligible effect on cell number (Fig. 2c).

Figure 2: Myc inhibition reduces proliferation and viability of V12Ha-Ras neuroprogenitors. (a) Neuroprogenitor cells freshly isolated from the subventricular zone (SVZ) of adult (3 months old) asymptomatic triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras control mice were grown as neurospheres in culture in serum-free medium supplemented with growth factors (EGF and FGF). Immunostaining for GFAP (green) and Ki67 (red) are shown. Nuclei were counterstained with Hoechst (blue). (b,c) NPG growth curves. Neuroprogenitors were plated as single cells in 12-well plates and treated with doxycycline (+Dox) or untreated (–Dox) for 3 days. Triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras cells expressing Omomyc show a significant reduction in the total number of cells after 3 days of treatment, compared with the untreated cultures. (P-value=0.0168, calculated by t-test; b). Notably, 3 days of Omomyc expression in control TRE-Omomyc;CMVrtTA does not affect significantly their growth rate (c). Data are shown as means±s.e. of triplicates (n=2). (d,e) Cell death was assessed by Trypan blue staining. Triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras NPGs have elevated numbers of dead cells after Omomyc expression for 3 days (d), whereas there is no difference in double transgenic TRE-Omomyc;CMVrtTA NPGs (e). Data are shown as mean+s.e. of triplicates. P-values were calculated by t-test. (f) BrdU staining of triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras and control TRE-Omomyc;CMVrtTA cells treated with doxycycline for 3 days or untreated. Data represent the mean of triplicates+s.e. (n=3). P-values were calculated by t-test. (g) Omomyc expression impairs the self-renewal ability of triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras NPGs. Single neuroprogenitor cells were plated at a clonal density (500 per well) in a 12-well plate and treated or not with doxycycline. After 21 days, the number of spheres was evaluated. Data are shown as means+s.e. of triplicates (n=2). P-values were calculated by t-test. Full size image

To determine the mechanism by which Omomyc expression blocks expansion of NPG cell cultures, we first examined cell death. Omomyc expression induced significant cell death in Ras-transformed NPGs but had no effect on the viability of control TRE-Omomyc;CMVrtTA cells (Fig. 2d,e). Omomyc also profoundly inhibited NPG proliferation: after 3 days of doxycycline treatment, we observed a significant reduction in the percentage of BrdU positive cells in TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras NPGs, down to a rate similar to that observed in TRE-Omomyc;CMVrtTA controls, in which no difference was observed as a consequence of Omomyc expression (Fig. 2f). Hence, Omomyc exerts its effect on Ras-transformed cells both by reducing proliferation and enhancing cell death.

Myc inhibition impairs NPG self-renewal capacity

According to the cancer stem cell hypothesis, the refractoriness of gliomas to eradication by chemo- and radiotherapy is due to a subpopulation of cells within the tumour that has stem cell features, including a high proliferation rate and self-renewal capacity. To investigate whether Myc inhibition impairs these characteristics, we employed a neurosphere self-renewal assay, measuring the number of cells able to reform a secondary neurosphere from a single cell. In this assay, NPGs are seeded at clonal density (500 single cells per well), then cultured with or without doxycycline for 21 days, at which point the number of spheres in each well is determined. Notably, Omomyc expression impairs the self-renewal ability of both the Ras-transformed and the control cells (Fig. 2g and Supplementary Fig. 1b), consistent with the crucial role ascribed to Myc proteins in promoting and maintaining self-renewal and stemness of both neoplastic and normal progenitor cells25,26.

Myc inhibition reverses the symptoms of GFAP-V12Ha-Ras mice

To test the therapeutic utility of Myc inhibition in advanced gliomas, we evaluated the impact of Omomyc expression in TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras mice presenting already clear neurological symptoms. Due to the variable tumour latency of our colony (see Fig. 1b, untreated–Omomyc animals), such symptoms appeared stochastically: the animals were initially hyperactive and often aggressive, but eventually became almost catatonic and unresponsive to external stimuli. At this point, they were treated with Omomyc. After 7 days of Myc inhibition, the therapeutic effect was dramatic. As documented by the movie (Supplementary Movie 1), mice were typically lethargic at the commencement of treatment, failed to groom and lacked appetite. After 7 days of Myc inhibition however, treated mice were active, responded again to external stimuli and were able to move, eat, drink and groom (Supplementary Movie 2).

Histological analysis of treated versus untreated samples clearly showed that untreated symptomatic mice exhibited regions with high density, GFAP-positive, pleomorphic and infiltrative astrocytes, typical histopathological features of human malignant astrocytomas (Fig. 3, middle versus upper panels). By contrast, the improved health of treated animals was associated with markedly reduced astrocytic (GFAP positive) cell density (Fig. 3, lower panels). Further immunohistochemical analysis of the residual GFAP-positive regions in Myc-inhibited symptomatic mice revealed a significant decrease in the number of proliferating cells compared with untreated mice (3.63% versus 7.83% positivity for the proliferation marker Ki67; Fig. 4a,b). In addition, we noted a small but significant increase in the number of apoptotic (TUNEL-positive) cells in Omomyc-expressing mouse brains when compared with Omomyc-negative controls (1.74% versus 0.48%; Fig. 4a,c), indicating that cell death contributes—at least to some degree—to the reduced cellular density observed upon Myc inhibition. A notable reduction in total cell number after Omomyc expression was confirmed by determining the number of nuclei present in five whole macroscopic fields (see total number of nuclei in treated versus untreated animals in Fig. 4b,c).

Figure 3: Histological analysis of symptomatic mice. Representative GFAP immunostaining of normal CMVrtTA and triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras brains. Left panels show that astrocytic density is reduced in Omomyc-expressing mice. Center panels focus on residual GFAP-positive regions. Right panels show higher magnification of astrocytes and active microglia. Full size image

Figure 4: Effects of Myc inhibition in the brain of GFAP-V12Ha-Ras symptomatic mice. (a) Symptomatic triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras mice were treated with doxycycline for 7 days (n=2) or untreated (n=3). Histological analysis (left panels) shows a reduction in Ki67 positive (proliferating) cells and an increase in dying TUNEL-positive cells in Omomyc-expressing mice. (b) Quantification of the Ki67 positivity from the histological analysis in a. The percentage of proliferating cells is shown in the graph, and the total number of cells counted in five fields is also indicated in the table. (c) Quantification of the TUNEL positivity from the histological analysis described above. The percentage of TUNEL-positive cells is shown in the graph, and the total number of cells counted in five fields is also indicated in the table. (d) Omomyc treatment causes the appearance of multinucleated cells. Symptomatic triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V12Ha-Ras mice were treated with doxycycline for 7 days. Histological analysis was performed on isolated brains to look for GFAP-positive astrocytes in tumour regions (green dye). Nuclei were counterstained with Hoechst (coloured pink here after merging with the GFAP images to facilitate visualization of the nuclei). (e) Quantification of the number of multinucleated GFAP-positive nuclei after Omomyc treatment (n=2) for 7 days compared with untreated mice (n=2). The average of two mice is shown+s.e. For each mouse, a minimum of 10 microscopic fields and 300 cells were scored. Data are shown as means+s.e. P-value was calculated by Student’s t-test. Full size image

Intriguingly, we observed that in the GFAP-positive regions remaining after Omomyc treatment, there was a significant increase in multinucleated GFAP-positive cells compared with untreated animals, suggestive of an effect of Myc inhibition on ploidy and mitosis of astrocytoma cells (Fig. 4d,e).

Myc inhibition triggers mitotic crisis in human GBM cells

To ascertain whether the antitumorigenic effect of Myc inhibition in the mouse model might translate into an analogous therapeutic impact in a human system, we investigated the effect of Omomyc expression in cell lines derived from human grade IV gliomas. U87MG cells27 were infected with retroviruses directing the expression of either GFP (control) or a GFP-Omomyc fusion protein. Consistent with our in vivo results in the GFAP-V12Ha-Ras mouse model, the total U87MG cell number was reduced on Omomyc induction, whereas GFP expression alone had no such effect (Fig. 5a). In addition, we observed a significant increase in cell death in U87MG cells expressing Omomyc, compared with control cells (Fig. 5b). Using a doxycycline-switchable lentiviral vector to drive expression of Omomyc, we also observed an increase in the proportion of flattened, senescent-like cells that stained positively for β-galactosidase (Fig. 5c). Broadly similar observations were made in Omomyc-expressing U373MG cells, which notably are mutant for p53 (Supplementary Fig. 2), although the cell flattening seen in U87MG cultures after Omomyc expression was not recapitulated (Supplementary Fig. 2c).

Figure 5: Omomyc causes growth arrest and increases cell death in U87MG cells. (a) Growth curves of U87MG infected with GFP control or GFP-Omomyc-expressing retroviruses. Total cell number was quantified at the indicated time points. Data are shown as the mean±s.e. of triplicates from a single experiment representative of three repeats. P-value at 3 days <0.05; P-value at 4 days <0.01, using a two-tailed t-test. (b) Cell death was assessed by Trypan blue staining. The number of dead cells was quantified at the indicated time points. Data are shown as mean±s.e. of triplicates. P-values were calculated by t-test. (c) U87MG cells were infected with a doxycycline-switchable lentiviral vector expressing Omomyc. Doxycycline treatment increases the number of β-galactosidase positive, senescent-like cells (left panels: typical β-galactosidase phase contrast images, right panel: quantification). The average of three experiments+s.d. is shown. P-values were calculated by a Student’s t-test. Full size image

Cytological examination of fixed Omomyc-expressing U87MG cells stained for tubulin and DNA showed that Omomyc expression causes a reduction in the mitotic index (0.96% versus 1.86% in control cells), but does not affect mitotic progression (the prophase+metaphase/anaphase+telophase ratio was 0.317 in Omomyc-expressing cells and 0.326 in GFP-expressing cells). A fraction of the dividing cells in Omomyc-expressing cultures (14.7%) displayed mitotic defects including multipolar spindles, aberrant chromosome segregation, chromatin bridges in ana-telophase and an irregular central spindle/midbody (Fig. 6a (control cells) versus Fig. 6b (Omomyc-expressing cells)). The final outcome of these defective mitoses was the formation of multinucleated and micronuclei-containing cells (13.6% after 72 h Omomyc expression; Fig. 6c,d, and Supplementary Fig. 3a). Similar results were obtained in U373MG cells with a doxycycline-switchable RFP-Omomyc-expressing vector (Supplementary Fig. 3b and c), where time-lapse imaging over the course of 72 h revealed that multinucleated cells enter mitosis but are unable to complete the mitotic process and may eventually die (Supplementary Movie 3).

Figure 6: Myc inhibition triggers micro- and multinucleation and mitotic abnormalities in U87MG cells. (a) Normal mitosis phases in control U87MG cells. Tubulin staining (second column and in red in the merged image) shows proper centrosome maturation and spindle assembly, whereas DAPI staining (first column and in blue in the merged image) highlights correct chromosome alignment and subsequent segregation. (b) Mitotic defects in Omomyc-expressing cells. The same tubulin and DAPI staining as above show that Omomyc induces defects in all mitotic phases, ranging from abnormal multipolar spindles (prophase and metaphase panels), to chromosome misalignments, segregation errors and lagging chromosomes (anaphase and cytokinesis panels), and finally absence of midbody and failed cytokinesis (bottom panel). Mitotic abnormalities involve 14.7% of mitotic figures in Omomyc-expressing cells. (c) GFP (green) immunofluorescence and DAPI nuclear counterstain (blue) show that after 24 h of GFP-Omomyc expression, U87MG cells become multinucleated (bottom panels) whereas that is observed much less frequently in control U87MG (top panel) that express GFP alone. (d) A representative image of a multinucleated Omomyc-expressing cell: α-tubulin staining (in red) shows that all nuclei belong to the same cell. The arrowhead indicates a typical micronucleus. Full size image

Myc inhibition alters SAE1 expression and PTP1B phosphorylation

Loss of SUMO activating enzyme (SAE) expression was recently found to exhibit synthetic lethality together with Myc overexpression28. One intriguing phenotypic outcome of SAE knockdown in Myc overexpressing tumour cells was the appearance of mitotic abnormalities and induction of mitotic catastrophe28. Interestingly, SAE1 is a direct target of Myc29. To verify whether SAE1 could be altered in response to Omomyc expression, we performed western blot analysis on GBM cell lines. We observed a clear reduction of SAE1 expression in U87MG (Fig. 7a). Hence, reduced SAE activity could be responsible for the aberrant nuclei and mitotic catastrophe observed in this cell line.

Figure 7: Omomyc causes alterations in proteins linked to mitotic cell death. (a) Western blot analysis of samples from GBM cells shows that Omomyc causes a decrease in SAE1 expression in U87MG but not U373MG cells. GBM cells with a lentiviral Omomyc expression cassette were treated with doxycycline for 6 days to induce Omomyc expression. Two independent sets of samples are shown for each of the GBM cell lines. (b) Western blot analysis of GBM samples shows that Omomyc expression triggers an increase in PTP1B phosphorylation in U373MG cells. GBM cells with a lentiviral Omomyc expression cassette were treated with doxycycline for 6 days to induce Omomyc expression. A Taxol control was used to indicate the typical phosphorylation-induced mobility shift of PTP1B during mitotic catastrophe. Taxol-treated control samples are nonadjacent bands from the same western blot. Full size image

On the other hand, in U373MG cells we observed no downregulation of SAE1 (Fig. 7a). Instead, phosphorylation of protein tyrosine phosphatase 1B (PTP1B)—a modification associated with and required for cell death during mitotic catastrophe30—was observed in response to Myc inhibition (Fig. 7b), to a degree similar to that caused by Taxol, a well-characterized microtubule stabilizer and mitosis inhibitor previously shown to cause PTP1B phosphorylation30.

These results offer at least two new potential mechanisms for Omomyc-induced cell death in GBM and hint at yet another aspect of proliferative biology in which Myc is involved.

Myc inhibition limits growth of patient-derived tumours

To determine whether the antitumorigenic capacity of Myc inhibition extends to primary human tumour samples and, consequently, might be clinically applicable in human glioma, we used a lentiviral vector to drive doxycycline-dependent Omomyc expression in patient-derived GBM neurosphere cultures derived from a surgically resected human GBM (Supplementary Fig. 4a). In culture, Myc inhibition for 2 weeks reduced the overall number of neurospheres (Fig. 8a) and inhibited self-renewal of spheres after their dissociation (Fig. 8b). No effect of doxycycline treatment was observed in neurospheres lacking the Omomyc lentiviral vector (Supplementary Fig. 4b).

Figure 8: Myc inhibition decreases the growth of patient-derived neurospheres in vitro and increases the survival of a xenograft mouse model. (a) Patient-derived neurosphere cultures derived from a resected human glioblastoma were infected with a lentiviral vector harbouring a doxycycline-regulatable Omomyc expression cassette. Myc inhibition by Omomyc decreases the number of spheres after 2 weeks of doxycycline treatment in culture. The mean+s.e.m. is shown (n=6). P-values were calculated by a Student’s t-test. (b) Omomyc expression in disaggregated neurospheres decreases their self-renewal ability. Neurospheres were dissociated into a single-cell suspension and 500 cells per well were added to a 96-well plate. The number of spheres was counted after 2–4 weeks in the presence or absence of doxycycline. The mean+s.e.m. is shown (n=5). P-values were calculated by a Student’s t-test. (c) A Kaplan–Meier survival curve of mice inoculated with patient-derived neurospheres and treated with doxycycline to trigger Omomyc expression (n=6), or with sucrose as the control (n=6). P=0.04 by log-rank test. (d) Omomyc expression increases the number of aberrant nuclei in nestin-positive cells. At the time of euthanasia, brains of the orthotopically injected mice described in 8c were fixed, and sections stained for nestin. Typical images are shown (green: nestin, pink: Hoechst). (e) Quantification of the experiment described in 8d. Five sections taken from different parts of each brain were used to score the number of aberrant nestin-positive nuclei (indicated by arrowheads). Untreated mice n=4, treated with doxycycline n=5. P-values were calculated by a Student’s t-test. Full size image

To investigate whether this potentially therapeutic effect might translate into an objective therapeutic impact, disaggregated neurospheres were orthotopically inoculated into the brains of NOD/SCID mice. Animals transplanted with neurospheres harbouring the Omomyc expression cassette were treated continuously with either doxycycline from 3 weeks after transplant (so that Omomyc was expressed throughout tumour development) or with sucrose in control mice. As expected, Myc inhibition significantly potentiated overall survival (Fig. 8c). Importantly, once again, we observed the appearance of aberrant nuclei in nestin-positive cells after Omomyc treatment (Fig. 8d,e).