The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that has a dual role in cancer, i.e., pro- or anti-tumorigenic, depending on the context. In medulloblastoma, the most frequent malignant pediatric brain tumor, several in vitro studies previously showed that AMPK suppresses tumor cell growth. The role of AMPK in this disease context remains to be tested in vivo. Here, we investigate loss of AMPKα2 in a genetically engineered mouse model of sonic hedgehog (SHH)-medulloblastoma. In contrast to previous reports, our study reveals that AMPKα2 KO impairs SHH medulloblastoma tumorigenesis. Moreover, we performed complementary molecular and genomic analyses that support the hypothesis of a pro-tumorigenic SHH/AMPK/CNBP axis in medulloblastoma. In conclusion, our observations further underline the context-dependent role of AMPK in cancer, and caution is warranted for the previously proposed hypothesis that AMPK agonists may have therapeutic benefits in medulloblastoma patients. Note: an abstract describing the project was previously submitted to the American Society for Investigative Pathology PISA 2018 conference and appears in The American Journal of Pathology (Volume 188, Issue 10, October 2018, Page 2433).

Medulloblastoma is the most common malignant brain tumor of childhood. Therapeutic approaches to medulloblastoma have led to significant improvements but are achieved at a high cost to quality of life. The Notch pathway governs cell proliferation in many biological contexts, including medulloblastoma tumorigenesis. Using our proteomic platform, we discovered an interaction between RBPJ, a key co-factor of Notch for the mediation of Notch signals, and L3MBTL3, a methyllysine reader for which deletions are observed in medulloblastoma. We demonstrated that L3MBTL3 is part of a molecular mechanism linking the KDM1A demethylase to Notch signal modulation. We hypothesize that malfunction of this molecular mechanism may contribute to the previously suggested tumor suppressor role of L3MBTL3 in medulloblastoma. In a survival analysis using our L3mbtl3 KO mouse in combination with a genetically engineered mouse model of medulloblastoma, we validated our hypothesis that L3mbtl3 is a tumor suppressor in this disease context. Our discovery provides insights into the role of the L3MBTL3 in medulloblastoma that could be harnessed in the future for the therapeutic benefit of medulloblastoma patients. Citation Format: Honglai Zhang, Ester Calvo Fernandez, Claire Peabody, Rork Kuick, Sung-Soo Park, Thomas Saunders, Sandra Camelo-Piragua, Jean-Francois M. Rual. Characterization of the role of L3MBTL3 in medulloblastoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3658.

The NOTCH/RBPJ pathway governs cell proliferation in many biological contexts, including SHH and Group#3medulloblastoma (MB) tumorigenesis. Using our proteomic platform, we discovered an interaction between RBPJ, a key co-factor of NOTCH for the modulation of the NOTCH/RBPJ signaling pathway, and L3MBTL3, a methyllysine reader. L3MBTL3 is recruited by RBPJ on chromatin at the enhancers of NOTCH/RBPJ target genes to repress their expression. Deletions of the L3MBTL3 locus are observed in patients with WNT and Group#3 MB and expression of L3MBTL3 in the SHH MB-derived cell DAOY inhibits cell growth, suggesting a putative tumor suppressor role for L3MBTL3 in MB. To further investigate the putative role of L3MBTL3 as a suppressor of MB tumorigenesis, we used our novel L3mbtl3 KO mouse in combination with a genetically engineered ND2:SmoA1 mouse model of SHH MB in a survival analysis. Furthermore, to identify the biological processes regulated by L3mbtl3 in MB, we analyzed by RNA-seq the transcriptome of L3mbtl3 KO mouse cerebella. Our survival analysis validated in vivo our hypothesis that L3mbtl3 is a tumor suppressor in this disease context. Indeed, our data show that [ND2:SmoA1; L3mbtl3+/-] mice have a significantly lower survival rate than ND2:SmoA1 mice (P = 0.032; Log-rank test). Moreover, our RNA-seq studies showed that L3MBTL3 regulates cell fate in the cerebellum via modulation the NOTCH/RBPJ signaling pathway. Hence, the RBPJ-L3MBTL3 interaction is at the heart of a molecular mechanism governing the repression of NOTCH/RBPJ target genes and malfunction of this molecular mechanism contributes to L3MBTL3’s tumor suppressor role in MB through aberrant “de-repression” of NOTCH/RBPJ target genes. Our discovery provides insights into the tumor suppressor role of the L3MBTL3 in MB that could be harnessed in the future for the therapeutic benefit of patients with MB.

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