Abstract 3656: The mTOR-activating GATOR2 complex is essential in PAX3-FOXO1-positive rhabdomyosarcoma
Authors: Sabnis Amit J. , Allegakoen David V. , Bivona Trever G. 
Published: August 2019

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, and can be genomically classified into fusion-positive and fusion-negative subsets. The former is driven by PAX3-FOXO1 or related chimeric transcription factors and has few other genetic alterations; the latter harbors recurrent mutations within the PI3K-RAS pathway. Despite these fundamental differences in cancer initiating events, current therapeutic approaches are identical between the two groups aside from chemotherapy intensification for some fusion-positive patients given their poorer outcomes.

Current pharmacologic strategies do not permit direct targeting of the PAX3-FOXO1 oncoprotein, so we undertook a CRISPRi genetic screen to identify genes whose loss was toxic to PAX3-FOXO1+ RMS cells, but tolerated in cells with knockdown of the fusion protein. We hypothesized that this would identify targetable dependencies created by PAX3-FOXO1 expression that might permit precision therapy for fusion positive RMS. Through this screen, we identified two genes within the mTOR-activating GATOR2 complex, MIOS and WDR24, as essential for the growth of PAX3-FOXO1 positive RMS cells.

We find that loss of MIOS is toxic to fusion-positive RMS cells due to a loss of mTORC1 signaling that normally initiates from signals of amino acid sufficiency. Restoration of mTORC1 activation by amino acids could be achieved by genetic suppression of either DEPDC5, a component of the RAG GAP GATOR1 complex that is normally inhibited by GATOR2, or of TSC1, which suppresses mTORC1 activity in the absence of PI3K signaling. Either genetic alteration completely rescued fusion positive RMS cells from the deleterious effects of MIOS loss. Correspondingly, we found that a fusion-negative RMS cell line with an activating NRAS mutation was insensitive to MIOS loss, suggesting that in this genetic context, amino acid signaling through GATOR2 is no longer necessary to activate mTORC1 and promote RMS survival.

In contrast, loss of WDR24 was toxic to both fusion positive and fusion negative RMS cells, and could not be rescued by genetic reactivation of mTORC1. Preliminary data demonstrate alterations in lysosome size and quantity after WDR24 suppression, adding to existing data in Drosophila that this gene plays a critical, mTOR-independent role in lysosome biogenesis, and showing that this role supports RMS survival.

As mTOR inhibitors are being tested in frontline phase 3 studies in North America, our work lends timely molecular insight into how the genetic basis of distinct RMS subtypes can influence the signals that promote mTORC1 function to support cancer cell survival. Based on our finding that amino acid activation of mTORC1 is a fusion-positive dependency, we are now testing combined mTOR inhibition with amino acid depletion as a precision therapeutic strategy in preclinical models of this aggressive childhood cancer.

Citation Format: Amit J. Sabnis, David V. Allegakoen, Trever G. Bivona. The mTOR-activating GATOR2 complex is essential in PAX3-FOXO1-positive rhabdomyosarcoma [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 3656.