The Abstract Book is now available for ARO in Baltimore 9-13 February 2019. Here is the entry for the Frequency Therapeutics / Will McLean presentation:
PD 67
Lgr5+ Cochlear Progenitor Cell Proliferation is Driven by the Combined Activation of the Wnt, NonCanonical Notch, and PI3K Pathways
Megan Harrison; Sara Strecker; Melissa Hill-Drzewi; Will McLean
Frequency Therapeutics
Loss of cochlear hair cells, which do not spontaneously regenerate in mammals, is a leading cause of hearing ARO Abstracts 361 Volume 42, 2019 loss. Previous work by McLean et al. 2017 showed that 3D organoid culture of Lgr5+ hair cell progenitors is a useful tool to study how various small molecules affect the ability of Lgr5+ cells to proliferate. In addition, 3D organoid culture can be a useful in vitro system for investigating the underlying biological pathways involved in progenitor cell regulation and plasticity. Enrichment for Lgr5+ cells can be achieved by treatment with molecules including the GSK3 inhibitor CHIR99021 (CHIR) and valproic acid (VPA). While CHIR is considered to promote proliferation by driving the Wnt pathway, the molecular mechanism by which VPA enhances CHIRdriven proliferation of Lgr5+ progenitor cells is not fully understood.
Here, organoid cultures were treated with CHIR and VPA individually as well as in combination, and the effects on gene modulation, total protein, and phosphorylated protein were assessed to elucidate the mechanism of synergistic Lgr5+ cell expansion. It was originally hypothesized that VPA functions by upregulating the Notch pathway. However, we find that VPA does not appear to lead to global gene or protein changes within the canonical Notch pathway, such as increased Hes and Hey expression. Exceptionally, Jagged1, a shared Wnt and Notch ligand, is upregulated by treatment with CHIR alone and is further upregulated upon dual treatment with CHIR and VPA, which correlates with the synergistic effects of these molecules in eliciting Lgr5+ cell proliferation. Engagement of Notch receptors by the addition of a peptide corresponding to the Jagged1 extracellular domain recapitulates the activity of VPA in Lgr5+ cell proliferation. Interestingly, inhibition of y-secretase, the enzyme that cleaves Notch receptors, does not affect Jagged1 induction level, signifying that Jagged1 induction may be y-secretase independent.
Together, these findings indicate that VPA may activate a non-canonical, y-secretase independent Notch pathway mechanism. One route of y-secretase-independent Notch signaling entails activation of the PI3K pathway. Indeed, we find that VPA activates the PI3K pathway in cultured Lgr5+ organoids as judged by increased levels of phosphorylated Akt. Further, a PTEN inhibitor, which activates the PI3K pathway, can substitute for VPA to enhance Lgr5+ cell proliferation and Jagged1 induction without inhibiting HDAC. Taken together, these findings indicate that combined activation of the Wnt, noncanonical Notch, and PI3K pathways elicits synergistic proliferation of Lgr5+ cochlear progenitor cells in culture.
https://cdn.ymaws.com/www.aro.org/resource/resmgr/mwm2019/2019_aro_mwm_abstracts_final.pdf
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