Capmatinib is an effective treatment for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy

Abstract

Background: Pediatric-type diffuse high-grade glioma (pHGG) is the most frequent malignant brain tumor in children and can be subclassified into multiple entities. Fusion genes activating the MET receptor tyrosine kinase often occur in infant-type hemispheric glioma (IHG) but also in other pHGG and are associated with devastating morbidity and mortality.

Methods: To identify new treatment options, we established and characterized two novel orthotopic mouse models harboring distinct MET fusions. These included an immunocompetent, murine allograft model and patient-derived orthotopic xenografts (PDOX) from a MET-fusion IHG patient who failed conventional therapy and targeted therapy with cabozantinib. With these models, we analyzed the efficacy and pharmacokinetic properties of three MET inhibitors, capmatinib, crizotinib and cabozantinib, alone or combined with radiotherapy.

Results: Capmatinib showed superior brain pharmacokinetic properties and greater in vitro and in vivo efficacy than cabozantinib or crizotinib in both models. The PDOX models recapitulated the poor efficacy of cabozantinib experienced by the patient. In contrast, capmatinib extended survival and induced long-term progression-free survival when combined with radiotherapy in two complementary mouse models. Capmatinib treatment increased radiation-induced DNA double-strand breaks and delayed their repair.

Conclusions: We comprehensively investigated the combination of MET inhibition and radiotherapy as a novel treatment option for MET-driven pHGG. Our seminal preclinical data package includes pharmacokinetic characterization, recapitulation of clinical outcomes, coinciding results from multiple complementing in vivo studies, and insights into molecular mechanism underlying increased efficacy. Taken together, we demonstrate the groundbreaking efficacy of capmatinib and radiation as a highly promising concept for future clinical trials.

Keywords: Capmatinib; Combination therapy; MET inhibition; Pediatric-type diffuse high-grade glioma; Preclinical trials; Radiosensitization.

Fig. 1

MET fusion IHG are large vascular tumors posing significant surgical challenges. a MRI images of IHG with CLIP2-MET fusion (right panel). Left panel: Left: T2 weighted image shows a large solid cystic tumor encompassing the entire right cerebral hemisphere, Middle: Subtraction weighted Image sequences (SWI). The yellow arrows indicate intra tumoral hemorrhagic regions. Right: T2 weighted image shows large tumor resection cavity after surgery. b MRI images of IHG with NPM1-MET fusion (right panel). Left panel: Left: T2 weighted MRI Image shows a large solid cystic tumor encompassing the entire temporal lobe of the left hemisphere. Right: Image post first attempt neuro-surgical resection. Due to massive bleeding and hemorrhage during surgery only a fraction of tumor could be resected. The yellow arrows show the large cysts within the tumor. c Images of IHG with HIP1-MET fusion (right panel). Left panel: Left: An emergent CT scan performed in the ER on a 4-week-old baby who presented with irritability and bulging anterior fontanelle. Shows a massive right hemispheric hemorrhagic tumor. The yellow arrow points toward the large hemorrhagic focus. Right: Diffusion Restricted images (DWI) of MRI. The restricted water diffusion (dark/black area noted by yellow arrow) represents high cellular density and proliferating tumor. d Histologic sections of a human MET-fusion tumor (TRIM24::MET) show large and abnormal thin-walled vessels invaded by the tumor cells (both upper panels), with mural thrombi (two left panels) and acute hemorrhages (second from left). Large areas of hemosiderin deposition, evidence of prior hemorrhages and hematoma, are noted in the tumor (second from right). Ample amounts of Gelfoam were needed to achieve hemostasis during surgery (far right). Scale bar is 150µm

Fig. 2

TFG-MET-driven mouse model and pharmacokinetic profiles of MET inhibitors. a Schematic illustrating the method and utilized vectors to induce CRISPR/Cas9-mediated Trp53 deletion and TFG-MET overexpression following in utero electroporations. b H&E staining and Immunohistochemical analysis of a tumor generated by in utero electroporation, visualized by the HA-tag of TFG-MET. In contrast to normal tissue (bottom right corners) tumors display elevated levels of pMET and pErk. Scale bars are 100 µm in large panel and 25 µm in high magnification inset. c, H&E staining showing a large and invasive HGG in the mouse brain. Red rectangle indicates the region shown in b. d H&E staining of a human MET-driven pHGG demonstrating similar features as murine neoplasms. Scale bars are 100 µm in large panel and 25 µm in high magnification inset. e Survival curve indicating penetrance and latency of tumors induces by in utero electroporation. f, Sanger sequencing of PCR products of the targeted Trp53 locus in a tumor revealed a 95bp deletion in all analyzed sequences (n=6). g, h Plasma(G)- and brain(H)-concentrations of capmatinib and crizotinib at the indicated time points after administration of CD-1 nude mice with the respective compounds. Three mice were analyzed per compound and time point. Error bars indicate the standard deviation. Dashed rectangles indicate time windows of radiation in the following preclinical allograft study

Fig. 3

Capmatinib is effective against TFG-MET-driven tumor cells. a Western blot of phosphorylated and total MET and the downstream effector Erk in cultured, murine tumor cells after different time points of crizotinib (cri) or capmatinib (cap) addition at the indicated concentrations. b Dose-response curves of murine tumor cells after treatment with capmatinib or crizotinib. Each dot represents one replicate of triplicates. Viable cells were analyzed 72 hours after compound addition using the CellTiter-Glo Assay. The vertical dotted lines indicate EC50 values. c Overview schematic depicting the various treatments and the two different cohorts of our preclinical allograft study. d Immunohistochemical stainings of phosphoproteins in tumors of the PD cohort, which were treated with the indicated therapies. Levels of pMET, pErk and pAkt were significantly reduced after capmatinib treatment. Scale bar is 50 µm. e Western blot of phosphorylated and total MET, Akt and Erk from allograft tumors treated with vehicle (veh), crizotinib or capmatinib alone or in combination with irradiation. f Quantification of luminescence signal of western blots in panel C normalized to the respective vehicle control. Each dot represents an individual replicate. Error bars display standard error of the mean. Statistical significance was determined using a One-Way ANOVA followed by Tukey’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

Fig. 4

Combining capmatinib and RT increases survival-rate and -time in vivo. a Kaplan–Meier curve of mice enrolled in the “Survival cohort”. All treatments started 1 week after transplantation. Radio therapy was administered for 6 days, delivering 12 Gy total. Compound treatment was continued for 84 days. After an additional 49 days of monitoring (140 days after transplantation) the trial ended and none of the remaining mice showed any hints of residual tumor. Three mice that were treated with capmatinib + RT reached this time point, whereas each of the other groups contained only 1 “survivor”. N = 8 (vehicle arms) or n = 10 (compound-treated arms), respectively. P-values for groups that displayed statistically significant survival differences are indicated. b Bioluminescence-imaging pictures from four representative mice of the vehicle arm (middle ranks according to initial luciferase intensity) and from all mice of the capmatinib + RT arm. First row is depicted in another intensity scale to visualize tumors in all mice. The depicted scale bar indicates the range from 5x10^5-1x10^7 photons/sec/cm²/sr. The combinatorial treatment induced tumor regression in 8/10 animals around day 21 on treatment. c Tumor burdens according to BLI of all enrolled mice before treatment are depicted as area of circles (left panel). The right panel shows the initial tumor sizes of mice that survived for 140 days without residual tumor. While the surviving animals of the vehicle groups displayed the smallest initial tumors, neoplasms of all sizes could be cured with combinatorial therapy of capmatinib and radiation. d Immunohistochemical analyzes of phosphoproteins in tumors of the Survival cohort, which were treated with the indicated therapies until onset of neurological symptoms. Phospho-MET, pErk and pAkt levels were significantly reduced in capmatinib-treated mice collected on days of treatment (Mo.-Fr.), however elevated levels reappeared in tissue collected during treatment pauses on weekends. Scale bar is 100µm

Fig. 5.

Sensitivity of human tumor samples to MET inhibition. a MRI images from an IHG patient with TRIM24-MET fusion. Left panel: Image at diagnosis showing a large solid cystic tumor filling the entire temporal lobe of the left hemisphere. Middle panel: Image at the end of resection and chemotherapy. Right panel: MRI image at recurrence. b The fusion encompassed TRIM24 exons 1-12 and exon 15 of c-MET, encoding a chimeric protein that contains the N-terminal moiety of TRIM24 and the c-MET kinase domain. c, tSNE projection of a combined methylation dataset comprised of a reference set of glioma subtypes (n=1128, circles from Capper, et al. Nature 2018, triangles from Clarke, et al. Cancer Discov 2020). The TRIM24-MET and TFG-MET tumor samples and cell lines from this study (squares, TRIM24-MET-i primary n = 4, cell culture n = 1; TRIM24-MET-r primary n = 3, cell culture n = 1; TFG-MET Models n = 6) group together with infant HGG with RTK fusion genes (IHG). d Dose-response curves of TRIM24-MET-i and TRIM24-MET-r cells after treatment with capmatinib, crizotinib or cabozantinib for 72 h. Data from three independent experiments. The vertical dotted lines indicate EC50 values

Fig. 6

Combination of capmatinib and RT eradicates human tumor cells in vivo. a Overview schematic depicting the four treatment arms of the preclinical study comparing in vivo response to capmatinib and cabozantinib. b Kaplan–Meier curve of mice enrolled in the study depicted in A. All treatments started 13 days after transplantation. Compound treatment was continued for 133 days. Within the subsequent 6 months of monitoring, 7 of 8 mice in the capmatinib-treated group experienced tumor relapse. c Overview schematic depicting the four treatment arms of the preclinical study comparing the combination treatment of capmatinib and RT vs either treatment alone. d Kaplan–Meier curve of mice enrolled in the study depicted in C. All treatments started 18 days after transplantation. Radiotherapy was administered at 0.5 Gy per day, delivering 10 Gy total. Compound treatment was continued for 301 days. After an additional 147 of monitoring, the trial ended with all mice having reached their tumor-induced or natural endpoint. e Trend of total flux (photons/sec/cm2/sr) at the cranial and spinal cord region of capmatinib-treated mice enrolled in the 4-arm preclinical trial depicted in C. f Bioluminescence-imaging pictures from mice of the vehicle + RT arm at the time closest to the humane endpoint and from capmatinib + RT treated mice at that time. Color scale range: 1.19x10^6-2.08x10^7 photons/sec/cm²/sr

Fig. 7

Capmatinib dysregulates expression of DNA repair genes and enhances radiation-induced DNA damage. a Expression of the indicated Mapk pathway signature (MPAS) genes in tumors of the PD cohort treated with vehicle (+/-RT) or capmatinib (+/- RT, focusing on the 4 strongly affected tumors by capmatinib-treatment, the 2 outliers were excluded for this analysis). With the exception of Epha4, expression of all analyzed Mapk pathway signature genes were inhibited in capmatinib-treated mice. Significantly downregulated (adj. p < 0.05) genes are in bold. b Heatmap of genes in the “DNA REPAIR_7” geneset (baderlab pathways 2019) demonstrating that capmatinib treatment leads to a reduced expression of DNA repair genes. c Correlation between total expression scores of the genesets “DNA REPAIR_7” and “CELL CYCLE_7” (baderlab pathways 2019) amongst all murine tumors (treated and untreated) analyzed by RNAseq in this study. Each dot represents one tumor. d Heatmaps showing expression of MAPK Pathway Activity Score (MPAS) genes for in vitro capmatinib treatments in cell lines derived from TRIM24-MET fusion tumors as compared to a DMSO vehicle control. Significantly downregulated (adj. p < 0.05) genes are in bold. e Western blots of RAD51 and β-ACTIN after indicated treatments of TRIM24-MET or TFG-MET cells for 24 hours. Capmatinib and crizotinib both induce downregulation of RAD51. f Western blots of MET and p-MET after indicated treatments of TRIM24-MET or TFG-MET cells for 24 hours, which serve as controls for Western Blots in e. g γH2AX-immunofluorescence staining of TRIM24-MET L97 human glioma cell lines at different recovery timepoints following 4 Gy-irradiation. Capmatinib (Cap)-treated cells display significantly higher levels of γH2AX compared to DMSO-treated (DMSO) cells. h quantification of γH2AX-foci in f. The percentage of cells with ≥20 γH2AX-foci is significantly higher in capmatinib-treated cells (black bar) compared to DMSO-treated cells (white bar) at 1, 2, 3 and 4 hours following irradiation. Error bars display standard error of mean, statistical significance was determined using t-test analysis. (****;p<0.0001, ***;p<0.001, **;p<0.01, *;p<0.05). Scale bar is 10µm. i Western blot of phosphorylated and total Kap1 from TFG-MET allograft tumors treated with vehicle (Veh), or capmatinib (Cap) alone or in combination with irradiation (RT). Samples 7-9 and 11-12 were collected 1 hr after RT, lane 10 was collected 3 hrs after RT, and shows time-dependent decrease of the DNA double-strand break signal. j Quantification of luminescence signal of western blots in panel h normalized to the vehicle control. Each dot represents an individual replicate. Error bars display standard error of the mean. Statistical significance was determined using a One-Way ANOVA followed by Tukey’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Lysate from lane 10 was excluded due to the different timepoint after RT