RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma

Abstract

27Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers 28 such as high-grade astrocytoma/glioblastoma, small cell lung cancer (SCLC), choroid plexus tumors and 29 small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional 30 compensation between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing 31 tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation of 32 rb1 and rbl1 by multiplex CRISPR/Cas9 genome editing in the true diploid Xenopus tropicalis to gain 33 insight into these in vivo compensatory mechanisms. We show that while rb1 inactivation is sufficient to 34 induce choroid plexus papilloma, combined rb1 and rbl1 inactivation is required and sufficient to drive 35 SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimicking 36 tp53 mutant X. tropicalis line, we demonstrate increased malignancy of retinoblastoma-mutant neural 37 malignancies upon concomitant inactivation of tp53. Interestingly, although clinical SC-PaNEC samples 38 are characterized by abnormal p53 expression or localization, in the current experimental models, the 39 tp53 status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential 40 for maintaining chromosomal stability. SCLC was only rarely observed in our experimental set-up, 41 indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 42 to delineate the tumor suppressor properties of Rbl1 and generate new insights in functional 43 compensation within the retinoblastoma protein family in suppressing pancreatic and specific neural 44 cancers. 45 Keywords 46 p107, p53, CRISPR/Cas9, Pancreatic neuroendocrine carcinoma, choroid plexus, retinoblastoma, Xenopus 47 tropicalis 48 49 50The interplay of the signaling networks controlling cell cycle (e.g. Retinoblastoma (RB)) and cell death 51 (e.g. p53) in suppressing the development of cancers including, amongst others, glioblastoma, choroid 52 plexus carcinoma, pancreatic neuroendocrine carcinoma and small-cell lung cancer was previously 53 demonstrated by clinical and animal modeling studies 1-6 . Unsatisfactory, the median survival prospects 54 for patients diagnosed with p53 and RB1 deficient cancers are extremely dismal, e.g. for small cell lung 55 cancer (SCLC) (stage IV: 8-10 months 7 ), small cell pancreatic neuroendocrine carcinoma (SC-PaNEC) (11 56 months 8 ), choroid plexus tumors (tp53 mutated WHO grade III: 2-4 months 9 ) and glioblastoma (12-18 57 months 10 ). As such, continued generation of novel and short latency preclinical models for these highly 58 aggressive cancers remains necessary to fuel rational design of molecular targeted drug therapies. 59 Intriguingly, the RB signaling network entails a family of three pocket proteins (RB1, RBL1, RBL2), 60 which in union tightly regulate G1/S cell cycle progression, and whose differential expression may 61 underl...

Engraftment of allotransplantated tumour cells in adult rag2 mutant Xenopus tropicalis

“…To assess transplantation potential in the novel rag2 -/line, a thymic tumour originating from an adult tp53 -/animal from a previous study (Naert, Dimitrakopoulou, et al, 2020) was isolated (Fig. 2A).…”

CRISPR-SID: identifying EZH2 as a druggable target for desmoid tumors viain vivodependency mapping

“…As a final extension to our CRISPR-NSID model, we performed triple multiplex parallel genome engineering of apc, ezh2 V659 and tp53 ( Table 2, Table S1d, S6c) 29 . Interestingly, all desmoid tumors (n=8) sampled in this cohort demonstrated biallelic tp53 editing, an event calculated as binomially unlikely (p<0.01) based on editing efficiencies of the tp53 gRNA (Table S6d).…”

“…Mdk, wisp-1, lox, adam-12, hmmr, nuak1, creb3l1, FAP-α, pclaf s8 , pclaf G10 , pycr1, Ezh2 R49 , Ezh2 S692 , Ezh2 V659 , tp53 and suz12 gRNAs were designed with the CRISPRScan software package 36 . tp53 gRNA was generated as described before 29 . In vitro transcription, purification and quality control of injection-ready gRNA was performed as described before (Table S7a) 11 .…”