Abstract B13: Targeting mTOR/JUN/AXL pathway in TSC-related tumors
Authors: Du Heng , Liu Heng-jia , Khabibullin Damir , Zarei Mahsa , Dreier John , Wu Chin-Lee , Henske Elizabeth , Kwiatkowski David 
Published: December 2020

Abstract
Renal angiomyolipoma (AML) and cysts are seen in 70-80% of patients with the human tumor suppressor gene syndrome tuberous sclerosis complex (TSC). Renal cell carcinoma (RCC) also is relatively common in TSC. Although two-hit loss of TSC1/2 is established in AML and TSC-RCC, the mechanisms and signaling pathways underlying TSC tumor development are not well understood. TSC-related tumor models were generated through i.p. tamoxifen (TM) injection (10mg/kg) at embryonic day 17.5 in Tsc2f/fROSA26-CreERT2 (n=24), Tsc2f/f CAG-CreERT2 mice (n=2) and Tsc1f/f CAG-CreERT2 mice (n=19). Extensive TSC-associated kidney papillary RCC (PRCC) and hybrid oncocytic/chromophobe tumors (HOCT) were seen in every kidney of these mice at age of 6 months, confirmed by IHC. A phospho-RTK immunoblot screen was performed using lysates from kidney tumors in comparison to normal kidneys. pAXL levels were ~2.8-fold higher versus normal kidney, whereas other pRTKs showed no apparent change. Immunoblotting confirmed the upregulated levels of both p-AXL (Tyr 702) and total AXL in kidney tumor lysates vs. normal kidneys. Q-RT-PCR of mRNA also showed 2-fold increase in AXL mRNA. Bioinformatic analysis suggested that AXL is a probable target of the transcription factor JUN. RNA-seq data showed that AMLs had the highest level of JUN expression in comparison to 28 different kinds of cancer (from TCGA). JUN was highly expressed in the mouse kidney tumors compared to normal kidneys in both protein and mRNA levels. JUN levels were also assessed using TSC-null cell lines (TSC2-/- angiomyolipoma cells (h), Tsc2-/- renal cell carcinoma cells (m), TSC1-/- bladder cancer cells (h), Tsc1-/- lung cancer cells (m)) versus their TSC add-back counterparts. All TSC-null cells showed a higher level of JUN and AXL compared to their TSC add-back counterparts. Both rapamycin (20nM) and Torin (500nM) treatment of these cell lines for 24h led to a reduction in JUN expression, with a stronger effect from Torin than rapamycin. To identify connections between JUN and AXL, JUN and AXL CRISPR KO were performed in 2 Tsc2-/- renal cell carcinoma cell lines. JUN KO led to a major decrease in AXL expression, and AXL KO led to a decrease of JNK, ERK and JUN. A positive feedback loop may exist between JUN and AXL through JNK and ERK pathway. R428, an inhibitor of AXL, was used to treat our Tsc2f/f ROSA26-CreERT2 mice and Tsc2+/- AJ mice (12.5mg/kg, twice daily, oral gavage, 3 weeks). Kidney tumor volume decreased dramatically compared with vehicle-treated mice in both cohorts (31.38 vs 4.11 mm³, p=0.001; 1.84 vs 0.05 mm³, p=0.02). These findings suggest that JUN and AXL are both upregulated in response to TSC1/TSC2 loss and mTORC1 activation and contribute to tumorigenesis, and that there is potential therapeutic value to targeting these events.
Citation Format: Heng Du, Heng-jia Liu, Damir Khabibullin, Mahsa Zarei, John Dreier, Chin-Lee Wu, Elizabeth Henske, David Kwiatkowski. Targeting mTOR/JUN/AXL pathway in TSC-related tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B13.