Abstract# LB201
MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically
Rebecca M. Hill1*, Sanne Kuijper2*, Janet C. Lindsey1*, Ed. C. Schwalbe1, Karen Barker2, Jessica K. R. Boult2, Daniel Williamson1, Zai Ahmad2, Albert Hallsworth2, Sarra L. Ryan1, Evon Poon2, Simon P. Robinson2, Ruth Ruddle2, Florence I. Raynaud2, Louise Howell2, Colin Kwok2, Abhijit Joshi3, Sarah Leigh Nicholson1, Stephen Crosier1, Stephen B. Wharton4, Keith Robson5, Antony Michalski6, Darren Hargrave6, Thomas S. Jacques7, Barry Pizer8, Simon Bailey1, Fredrik J. Swartling9, Kevin Petrie2*, William A. Weiss10,11, Louis Chesler2&#, Steven. C. Clifford1&#. 1Newcastle University, Newcastle upon Tyne, U.K.; 2The Institute of Cancer Research, Sutton, U.K.; 3Royal Victoria Infirmary, Newcastle upon Tyne, U.K.; 4University of Sheffield, Sheffield, U.K.; 5Children’s Brain Tumour
Research Centre, University of Nottingham, Nottingham, U.K.; 6Great Ormond Street Hospital for Children, London, U.K.; 7UCL Institute of Child Health/Great Ormond Street Hospital for Children, London, U.K.; 8Alder Hey Children’s Hospital, Liverpool, U.K.; 9Uppsala University, Uppsala, Sweden.;10Helen Diller Family Comprehensive Cancer Center and 11Brain Tumor Research Center, University of California, San Francisco, California, U.S.A.
Abstract There are currently no effective therapies for children with relapsed medulloblastoma; over 95% of patients die, accounting for ~10% of childhood cancer deaths. While the clinical and biological features of medulloblastoma are increasingly well understood at diagnosis, management following relapse typically focusses on quality of remaining life, with biopsy rarely performed and little biological data currently available to inform effective treatments. We therefore undertook comprehensive investigations of 29 relapsed medulloblastomas and contrasted these with their diagnostic counterparts. At diagnosis, medulloblastoma is heterogeneous comprising four molecular subgroups (WNT (MBWNT), SHH (MBSHH), Group 3 (MBGroup3) and Group 4 (MBGroup4)) with distinct clinical, pathological and molecular features. At relapse, subgroup was unchanged but other clinical and biological characteristics were commonly altered. Most notably, MYC gene family (MYC or MYCN) amplifications and P53 pathway defects commonly emerged in combination at relapse following standard upfront radiotherapy and chemotherapy (7/22, 32%). This group of patients all died quickly within 9 months following relapse (0.57 years (0.33–0.72 years range) median time to death postrelapse, versus 1.22 years for other tumors (0.02–2.9 years); P = 0.0165, log-rank test). Spontaneous development of Trp53 inactivating mutations was similarly common in a transgenic model of MYCN driven medulloblastoma (GTML;Glt1–tTA/TRE–MYCN–Luc). Direct modelling of this interaction in GTML/Trp53KI/KI mice completely deficient for p53-enhanced medulloblastoma formation, mimicking clinical and pathological characteristics of relapsed human tumors characterized by this genotype. Therapeutic inhibition of Aurora-A kinase in this model promoted degradation of MYCN, reduced tumor growth and prolonged survival. In summary, medulloblastomas are characterized by unique biological features at relapse. The identification of P53– MYC interaction as a biomarker of clinically aggressive disease that may be targeted therapeutically indicates the necessity for routine biopsy at relapse to direct palliative care and the development of targeted treatments.
Results
Figure 2. Combined P53 pathway defects and MYC/MYCN amplification commonly emerge following standard upfront radiotherapy and chemotherapy and correlate with rapid disease progression after relapse. Analysis of (a) association, (b) frequency of
Figure 1. Relapsed medulloblastomas maintain molecular subgroup but are enriched for multiple high-risk clinical and molecular features. (a) Consensus clustering (left) and principal component analysis (PCA) (right) of medulloblastoma subgroups at diagnosis and relapse. Consensus molecular subgroups: red, MBSHH; blue, MBWNT; yellow, MBGroup3; green, MBGroup4. In the PCA plot, subgroups assigned at diagnosis are represented by circles and those assigned at relapse by squares. (b) Frequency of high-risk disease features within the present paired relapse study cohort sampled at diagnosis and relapse, compared to large historic cohorts sampled at disease diagnosis. (P, Fisher’s exact test.) (c) Acquisition of molecular and clinical disease features between diagnosis (top) and relapse (bottom). Left to right; P53 protein accumulation, TP53 homozygous missense mutation (Pro152Leu), MYCN amplification (green versus centromeric control (red)), LCA acquisition and metastatic spread.
occurrence and (c) distribution within molecular subgroups of combined P53 pathway defects and MYC/MYCN amplification at diagnosis and relapse. (d) Survival of patients with tumors harboring combined P53–MYC gene family defects versus other tumors, showing time from diagnosis to relapse (left) and relapse to death (right). P, Log rank test, Bonferroni corrected. (e) Detailed clinical, pathological and molecular demographics of patients with combined P53–MYC family gene defects at relapse. Consensus molecular subgroup (red, MBSHH; blue, MBWNT; yellow, MBGroup3; green, MBGroup4). Pathology variant (CLA, classic; LCA, large-cell/anaplasia; DN, nodular/desmoplastic; NOS, medulloblastoma not otherwise specified). Disease location (local, M0/M1; distant, M2+) current status (DOD, died of disease). Chromosome 17 status (red, loss; green, gain). D, diagnosis; R, relapse. Feature present, grey square; feature absent, white square; data not available, cross-hatching.
Figure 3. Aberrant expression of MYCN in combination with Trp53 loss of function drives highly penetrant and aggressive medulloblastoma. (a) Kaplan-Meier survival curves for GTML/Trp53KI/KI (n = 43), GTML/Trp53KI/WT (n = 83) or GTML transgenic mice (n = 50) mice as indicated. P, Log rank test. (b) H&E (Hematoxylin and eosin) and immunohistochemical staining indicating levels of MYCN protein and cell proliferation (Ki-67) in GTML/Trp53KI/KI and GTML transgenic mice. Scale bars represent 50 µm. (c) Results of a sub-group classification using a SVM (Support Vector Machine) trained on human medulloblastoma expression profiles and tested on mouse expression profiles. Cross-species projection is performed using NMF (Non-negative Matrix Factorization) and demonstrates that murine MYC and MYCN-GTML tumors recapitulate the expression profiles of Human MBGroup3. (d) Kaplan-Meier survival for GTML/Trp53KI/KI mice treated with doxycycline (dox, 200 mg kg–1, n = 4) or tamoxifen (4-OHT, 1 mg kg–1, n = 4) compared to vehicle (n = 3) as indicated. P, Log-rank test. (e) GTML/Trp53KI/KI mice, which co-express firefly luciferase (FLuc) were treated with 4-OHT, dox or vehicle for 9 days. Bioluminescent imaging of GTML/Trp53KI/KI mice after 9-days treatment with dox or 4-OHT as indicated (top). Luminescence intensity at day 0 and day 9 are shown (bottom). Data points represent individual mice. P, unpaired t-test. (f) H&E and immunohistochemical staining indicating levels of MYCN, Ki-67 or apoptosis (Cleaved caspase 3) in GTML/Trp53KI/KI mice after treatment with dox or 4-OHT. Scale bars represent 50 µm. (g) RNAscope 2plex chromogenic assay was performed for Cdkn1a (red) on brain sections from GTML/Trp53KI/KI mice treated with either 4-OHT or vehicle control. Samples were co-stained for expression of the Ubc (ubiquitin C) housekeeping gene (green). The positive control used probe sets directed against Ppib (Peptidylprolyl Isomerase B, Cyclophilin B) (red) and Polr2a (DNA-directed RNA polymerase II subunit RPB1) (green). The negative control used probe sets directed against the dapB (dihydrodipicolinate) reductase gene from B. subtilis. Sections were counterstained with Gill’s Hematoxylin. (h) Fold difference of human MYCN or mouse Cdkn1a RNA levels in tumor tissues treated with either 4-OHT or dox. (P, unpaired t-test.)
Author information Email address of corresponding authors:
[email protected],
[email protected] *Contributed equally to this work and are co-first authors, &Contributed equally to this work and are co-senior authors, #Joint corresponding authors.
Figure 4. Therapeutic targeting of the MYCN/Aurora-A interaction inhibits tumor growth and prolongs survival in GTML/Trp53KI/KI mice (a) Proximity ligation assay (PLA)
analyzing MYCN/Aurora-A complexes in GTML/Trp53KI/KI neurospheres. Left panel shows close proximity (< 40 nm) of antibody conjugated PLA probes that have been ligated, amplified and detected with complementary fluorescent probes. Red dots represent the presence of MYCN or Aurora-A protein, or MYCN/Aurora-A interactions as indicated. Scale bars indicate 20 μm. Where indicated, MLN8237 was added for 48 hr. Right panel shows mean values of signals (red dots) per cell representing MYCN expression or MYCN/Aurora-A interactions. Values are derived from triplicate biological replicates and error bars represent standard deviations. P, unpaired t-test. (b) Kaplan-Meier survival for GTML/Trp53KI/KI mice treated with MLN8237 (30 mg kg–1, n = 6), GDC-0449 (50 mg kg–1, n = 4) or vehicle (n = 7) as indicated. (P, Log rank test.) (c) Longitudinal bioluminescent (firefly luciferase) imaging of GTML/Trp53KI/KI mice shows stable firefly luciferase expression after treatment with MLN8237 (50 mg kg–1) compared to vehicle. (d) Longitudinal MRI analysis of tumor volume on the axial plane (top). Representative MRI images of the axial plane show a mean increase of MLN8237-treated animals compared to vehicle as indicated at day 0 and last day of treatment (bottom). (e) H&E and immunohistochemical staining indicating levels of MYCN protein, cell proliferation (Ki-67), apoptosis (Cleaved caspase 3), or mitotic activity as measured by phosphorylated Ser10 on histone H3 (H3 p-S10) after treatment with GDC-0449 or MLN8237. Scale bars represent 50 µm. (f) Immunoblotting of MYCN protein levels, and total and phosphorylated Thr288 on Aurora-A (p-T288 Aurora-A) in MLN8237-treated tumor tissues. For (e) and (f) animals were treated with vehicle, GDC-0449 or MLN8237 for 48 hrs and samples taken 2 hrs after final administration of agent.
