Poster Presentation 5th International Symposium on Phaeochromocytoma and Paraganglioma 2017

Novel germline MAX mutations impair nuclear localisation (#82)

Edward Kim 1 2 , Diana (Dindy) E Benn 1 2 , Anne Louise Richardson 1 2 , Catherine Luxford 1 , Margaret Zacharin 3 , Emma Duncan 4 , Trish Dwight 1 2 , Roderick J Clifton-Bligh 1 2
  1. Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, NSW, Australia
  2. University of Sydney, Sydney, NSW, Australia
  3. Department of Endocrinology and Diabetes, Royal Children's Hospital, Melbourne, VIC, Australia
  4. Department of Endocrinology, Royal Brisbane Hospital, Brisbane, QLD, Australia

Background: MYC-associated factor X (MAX) is a key protein involved in maintaining balance of cell differentiation, proliferation and apoptosis. Primarily expressed in the nucleus, functional MAX forms a heterodimer with MYC or MXD to regulate transcriptional activity, disruption of which has been reported in various neoplasias. Germline mutations in the gene encoding MAX have been associated with the development of hereditary phaeochromocytoma and paraganglioma (PC/PGL). We recently identified two novel germline MAX (NM_002382) mutations, p.Leu64Pro and p.Ala67Asp. 

Objective: The aim of this study was to assess the biological impact of novel MAX mutations, specifically their effect on cellular localisation.

Method: Human embryonic kidney cells (HEK293) and rat derived PC cells (PC12) were transfected with DDK-tagged wild type MAX (pCMV6-AC-MAXwt-DDK) or mutant MAX constructs (generated by site-directed mutagenesis). The pathogenic MAX mutant p.Met74Val was used as a positive control. Western blot analyses were used to compare DDK expression between mutant MAX and wild type MAX in the cytosolic and nuclear fractions (enriched using a cellular fractioning kit).  Expression of DDK-MAX was normalised to the cytosolic marker (alpha-Tubulin) or nuclear marker (Lamin B1).

Results: Protein expression of DDK-tagged wildtype MAX was shown to be predominantly found in the nucleus, as expected. Expression of DDK-MAX was significantly elevated within the cytoplasm in the MAX mutants, p.Leu64Pro and p.Met74Val, when compared to wild type (n=3, p<0.05). Conversely, in the nuclear compartment expression of the MAX mutants, p.Leu64Pro, p.Ala67Asp and p.Met74Val, were significantly reduced compared to wild type (n=3, p<0.05).

Conclusion: Novel MAX germline mutations, p.Leu64Pro and p.Ala67Asp, discovered in Australian PC/PGL cases show impaired nuclear localisation when compared to wild type MAX. Further exploration of the mechanism(s) associated with MAX mutants in PC/PGL may uncover potential strategies for targeted therapy.