# Deciphering the Impact of AKT1 Pathogenic Variants in Juvenile Granulosa Cell Tumors Using a Drosophila Model

## TL;DR

This study uses fruit flies to show how harmful AKT1 gene changes cause abnormal cell growth and signaling in juvenile granulosa cell tumors.

## Contribution

The study reveals both gain-of-function and loss-of-function effects of AKT1 variants in a Drosophila model of JGCTs.

## Key findings

- Mutant AKT1 variants caused increased cell size and polarity defects in Drosophila wings and ovarian cells.
- Transcriptomic and proteomic analyses showed distinct gene and protein changes linked to mutant AKT1 activity.
- Network analysis highlighted AKT1 as a central driver of tumorigenic signaling in JGCTs.

## Abstract

Juvenile-type granulosa cell tumors (JGCTs) manifest during the prepubertal period as precocious pseudo-puberty and/or dysmenorrhea. We have previously identified pathogenic variants in AKT1 in JGCTs. This study aims to understand how these variants affect cellular function at the phenotypic and molecular levels using a Drosophila model. Transgenic Drosophila models expressing WT AKT1 and four pathogenic variants were created under the control of tissue-specific promoters. Phenotypic effects were studied by assessing Drosophila wings for cell division and growth using wing surface and trichome density and ovarian follicular cells were examined for subcellular localization and morphology. Molecular analyses included mass spectrometry to identify differentially expressed proteins (DEPs) and phospho-peptides, along with RNA-Seq to characterize transcriptomic changes. Wings expressing mutated AKT1 showed increased surface area and reduced trichome density, indicating larger cells. In ovarian follicular cells, WT AKT1 localized primarily to the cytoplasm, while mutated AKT1 variants were associated with the plasma membrane, leading to morphological abnormalities and increased cell size. Mass spectrometry revealed numerous DEPs and phospho-peptides, highlighting changes in pathways such as glycolysis and Rho GTPase signaling. Transcriptomics demonstrated a clear gain of function for mutated AKT1 in activating a subset of genes. However, several genes upregulated by WT AKT1 were less effectively activated by the mutants, indicating a potential loss-of-function in transcriptional regulation for this subset, revealing an unexpected mechanistic complexity. Network analysis of interactions involving DEPs, phosphorylated proteins, and transcription factors suggests these elements mediate the observed proteomic and transcriptional alterations. Taken together, the results underscore the utility of Drosophila models in unraveling the biological relevance of AKT1 pathogenic variants in cancer.

•Pathogenic AKT1 variants found in juvenile granulosa tumors were introduced into a Drosophila model.•AKT1 variants cause abnormal cell growth, polarity defects and morphological alterations in tissues.•Mass spectrometry revealed specific proteomic signatures associated with mutant AKT1 activity.•Transcriptomics revealed unexpected gain-of-function and loss-of-function effects for distinct gene sets.•Network analysis linking DEPs, phosphoproteins, and TFs, highlight AKT1 as key cancer driver.

Pathogenic AKT1 variants found in juvenile granulosa tumors were introduced into a Drosophila model.

AKT1 variants cause abnormal cell growth, polarity defects and morphological alterations in tissues.

Mass spectrometry revealed specific proteomic signatures associated with mutant AKT1 activity.

Transcriptomics revealed unexpected gain-of-function and loss-of-function effects for distinct gene sets.

Network analysis linking DEPs, phosphoproteins, and TFs, highlight AKT1 as key cancer driver.

We use transgenic Drosophila to investigate how pathogenic AKT1 variants, identified in Juvenile granulosa cell tumors, impact cellular behavior. Combining in vivo phenotypic analysis with mass spectrometry and RNA-Seq, we reveal that AKT1 variants promote abnormal growth and polarity defects, accompanied by distinct proteomic and transcriptomic changes. These findings highlight the complexity of oncogenic AKT1 signaling and underscore the value of Drosophila models for dissecting tumorigenic mechanisms.