Inhibition of the amino‐acid transporter LAT1 demonstrates anti‐neoplastic activity in medulloblastoma

Abstract Most cases of medulloblastoma (MB) occur in young children. While the overall survival rate can be relatively high, current treatments combining surgery, chemo‐ and radiotherapy are very destructive for patient development and quality of life. Moreover, aggressive forms and recurrences of MB cannot be controlled by classical therapies. Therefore, new therapeutic approaches yielding good efficacy and low toxicity for healthy tissues are required to improve patient outcome. Cancer cells sustain their proliferation by optimizing their nutrient uptake capacities. The L‐type amino acid transporter 1 (LAT1) is an essential amino acid carrier overexpressed in aggressive human cancers that was described as a potential therapeutic target. In this study, we investigated the therapeutic potential of JPH203, a LAT1‐specific pharmacological inhibitor, on two independent MB cell lines belonging to subgroups 3 (HD‐MB03) and Shh (DAOY). We show that while displaying low toxicity towards normal cerebral cells, JPH203 disrupts AA homeostasis, mTORC1 activity, proliferation and survival in MB cells. Moreover, we demonstrate that a long‐term treatment with JPH203 does not lead to resistance in MB cells. Therefore, this study suggests that targeting LAT1 with JPH203 is a promising therapeutic approach for MB treatment.

and chemotherapy. Shh and Wnt subgroups show the highest overall survival while group 4 and, to a higher extent, group 3 MBs are of poor prognosis with a strong tendency to form metastasis. 2,3 Although the overall survival at 5 years reaches 70% in all MB types taken together, even peaking over 90% in the Wnt group, toxic effects due to the treatments often lead to irreversible damages that severely hamper the children's cognitive development and general quality of life. In particular, RT dose and age of exposure to RT are well-documented risk factors for long-term cognitive impairments in MB survivors. [7][8][9] Reducing RT doses has been proposed; however, it is unsuitable for high-risk MBs. 8,9 Moreover, recurrences are fatal in a large majority of cases and are characterized by strong divergence of the relapsed tumours as compared to the initial tumours. 10 A challenge therefore lies in finding new treatments that combine high efficacy and little toxicity. Personalized targeted therapy appears to be a promising approach in this context. 11,12 Clinical trials involving Shh pathway targeted inhibitors have enrolled Shh group MB patients based on this assertion. To date, these trials have not led to the expected results as they have induced unbearable toxicity and tumour resistance to the treatment. [13][14][15] All these features support the need to develop and design novel therapies against alternative targets.
Rapidly growing tumours experience an increased demand for nutrients in order to sustain their proliferative metabolism. Up-regulation of processes related to the supply of glucose, amino acids, and lipids is thus a hallmark of cancer metabolism. 16,17 Essential amino acids (EAAs), which cannot be synthetized de novo by human cells, are absolutely required for cancer cell proliferation. Indeed, some of these nutrients can also be converted to essential metabolite intermediates of the TCA cycle, further participating in tumour energy metabolism and macromolecule synthesis. Notably, catabolism of the branched chain EAA subclass (leucine, isoleucine and valine) is required for growth of some brain tumours. 18 In addition, leucine is an essential signalling molecule required to sustain the activation of mTORC1, the master kinase of protein, lipid, nucleotide syntheses and cell proliferation. 19,20 Therefore, considering the high expression of LAT1 in MB cells, 21 we have suggested that targeting EAA uptake might be an innovative strategy for MB treatment. The L-type amino acid transporter 1, LAT1 (SLC7A5), is a 12-transmembrane protein responsible for Na + -independent transport of large neutral EAA (Leu, Val, Ile, Phe, Trp, His, Met, Tyr) in tight association with chaperone CD98 (SLC3A2). LAT1 is an obligatory exchanger with the uptake of one amino acid (AA) being coupled to the efflux of another. LAT1 is overexpressed in aggressive human cancers including MB and has been described as a potential therapeutic target. 21,22 In a previous study, we demonstrated that LAT1 activity is essential for tumour growth. 23 Indeed, genetic disruption of LAT1 in colorectal and lung adenocarcinoma cell lines leads to EAA starvation, mTORC1 inactivation and growth arrest. 23,24 In addition, our lab with others demonstrated that treatment with JPH203, a specific inhibitor that targets LAT1 and no other LAT, recapitulates these effects in several cancer cell lines, including glioma cell lines. 23,25,26 However, the efficiency of this compound had never been addressed in MB. In this study, we demonstrate that LAT1 is the main leucine transporter in two independent cell lines isolated from Shh (DAOY) and Group 3 (HD-MB03) MBs. 27,28 We subsequently show that JPH203 treatment disrupts AA homeostasis, mTORC1 activity as well as proliferation and survival of both MB cell lines. Furthermore, in contrast to its effect on MB cell lines, JPH203 displays low toxicity on two normal cerebral cell types. Finally, we show that although MB cells try to adapt to a JPH203 chronic treatment by up-regulating the expression of AA transporters, no resistant clones could be isolated from the cell populations. Altogether our in vitro results strongly suggest that JPH203 is a promising therapeutic candidate for treating MB.

| Cell culture and pharmacological inhibitor
The murine cerebellar astrocyte cells C8-D1A were obtained from the and cultivated in Minimum Essential Medium (MEM, Invitrogen) supplemented with 10% iFCS and 90% RPMI-1640 supplemented with 10% iFCS respectively. Primary murine neurons were isolated from E16.5 mouse embryos cortices dissociated with trypsin as previously described. 29 Briefly, neurons were plated onto 12-well plates and cul-
Quantification of the intensity of the Western blot bands were performed with ImageJ software (NIH, USA).

| Proliferation and viability assays
The different cell lines (2.5 × 10 4 cells for 7 days, 5 × 10 4 cells for 3 days) were seeded onto 6-well plates in triplicates. We measured proliferation by trypsinizing the cells and counting them daily

| Three-dimensional growth assay
DAOY and HD-MB03 3-D cultures were prepared using ultra-low attachment 96-well plates (Corning). Cells (5000) were seeded in 200 µL medium per well. The spheroids were cultured for 8 days and pictures were taken with an AMG Evos microscope 40x objective (Thermo Fisher Scientific Inc). Spheroid areas were measured using ImageJ 1.51j8 software (National Institute of Health). 30

| Scratch assay
DAOY cells (800 000) were seeded in 60 mm diameters and grew until they reached confluency (2 days). A wound was then done in the monolayer by scratching it with a 10 µL pipette tip (Starlab).
The culture medium was then replaced by fresh medium containing DMSO or the indicated concentrations of JPH203. Paired measurements of the wound width were done at 0 and 8 hours at six different places for each condition.

| Statistical analysis
Data are expressed as mean ± SD. Each experiment was performed at least three times. Statistical analysis was done with the unpaired Student's t test. Differences between groups were considered statistically significant when P < 0.05 in a Student's t test.

| LAT1 is the main Na + independent leucine transporter in HD-MB03 and DAOY MB cell lines and is essential for AA homeostasis and mTORC1 activity
We first demonstrated that LAT1 and its chaperone CD98 are expressed in HD-MB03 and DAOY cell lines ( Figure 1A). The multiple bands observed in the CD98 blot are due to post-translational modifications of the protein. None of them is detectable in protein extract obtained from CD98 knock-out cells. 23 Functional activity of LAT1 was quantified by measuring the Na + -independent rate of leucine transport in the presence or absence of JPH203, a specific LAT1 inhibitor ( Figure 1B). JPH203 completely abolished leucine uptake ( Figure 1B), suggesting that LAT1 is the main functional Na + independent leucine transporter in these two MB cell lines. Next, we investigated the effects of LAT1 inhibition on the two AA-sensing pathways: GCN2 and mTORC1 ( Figure 1C). 31 In both cell lines, LAT1 inhibition resulted in the activation of the AA stress response pathway GCN2, observed through increased phosphorylation of GCN2 and EIF2α and up-regulation of ATF4 expression ( Figure 1C and Figure S1). Moreover, JPH203 treatment resulted in a strong decrease in mTORC1 activity, scored by the phosphorylation of its two effectors: p70-S6K1 and the ribosomal protein S6 ( Figure 1C and Figure S1). Altogether these results demonstrate that JPH203 treatment leads to AA starvation and suggest that LAT1 activity is required for AA homeostasis in cells belonging to different MB subgroups. Finally, we tested the effect of LAT1 on the migration capacities of MB cells using a scratch assay on a confluent layer of DAOY cells.

| Pharmacological inhibition of LAT1 impairs MB cell proliferation, survival and migration abilities
JPH203 treatment resulted in a 25% reduction in scratch closure after 8 hours compared to cells treated with DMSO ( Figure 3A,B).
This suggests that LAT1 activity is required to sustain MB cell motility. Altogether, these findings demonstrate that LAT1 through its EAA transport activity promotes some of the key malignant features of MB cells while bearing seemingly no or low toxicity towards healthy cerebral cells.

| Chronic treatment of MB cells with JPH203 induces cellular adaptation but no resistance
In order to test the development of resistance mechanisms, HD-MB03 and DAOY cell lines were incubated for more than 120 days in JPH203-supplemented medium (20 and 30 µmol/L). The levels of LAT1 and its chaperone CD98 were dramatically increased in F I G U R E 1 L-type amino acid transporter 1 (LAT1) is the main leucine Na + independent transporter expressed in MB cell lines HD-MB03 and DAOY and is essential for AA homeostasis and mTORC1 activity. A, Western blot analysis of the expression levels of LAT1 and its chaperone CD98 in HD-MB03 and DAOY. Tubulin served as a loading control. The results presented are representative of at least three independent experiments. B, Transport assay using radio-labelled leucine ( 14 C-LEU) in the absence or presence of 10 µmol/L of JPH203 (***P < 0.001, Student's t test). C, Activity of the two AA sensing pathways GCN2 and mTORC1 were analysed by immunoblot in the absence or presence of either 20 or 30 µmol/L of JPH203. ERK1/2 served as a loading control (the experiment presented here is representative of at least three independent experiments) response to chronic exposure to JPH203 ( Figure 4A). Consistently, the mRNA levels of LAT1 and LAT3 were also increased ( Figure S1).
Nonetheless, JPH203 still displayed strong anti-proliferative and cytotoxic effects on both cell lines ( Figure 4B,C). These results strongly suggest that this 4-month adaptation was insufficient to induce resistance to LAT1 inhibition although the chronic treatment with JPH203 led to up-regulation of some components of the amino acid transport machinery.

| D ISCUSS I ON
The most aggressive forms of MBs as well as local or metastatic relapses generally show a high degree of resistance to classical treatments. It is therefore crucial to discover new treatments presenting important properties such as selective toxicity towards cancer cells and low toxicity towards normal tissues. JPH203 is a tyrosine analogue that inhibits LAT1 with a high specificity as compared to other compounds. 22,26 The proposed mode of action of this compound is via competition with other substrates of the LAT1 transporters. 22 In this study, we show that JPH203-in- this study as well as other models mostly rely on LAT1 ( Figure 1B). 23 Since leucine is an essential amino-acid, these cells cannot grow in the absence of LAT1 activity. Although our results suggest a low toxicity of JPH203 towards normal cells, it is important to mention that the impairment of the transport of branched-chain amino acids at the blood-brain barrier caused by a LAT1 deficiency has also been described to cause autism spectrum disorders in mice. 33  The transmembrane transport of JPH203 was demonstrated to be mediated by transporters of the organic-anion-transporting peptides (OATP, SLCO) and the organic anion transporters (OAT, SLC22) families, namely OATP1B1, OATP1B3, OATP2B1 and OAT3. 35 Interestingly, some of these transporters were also described to be expressed in brain capillary endothelial cells constitutive of the blood-brain barrier (BBR). 36 This suggests that a route for JPH203 to cross the BBR could be provided by transmembrane transporters, making brain tumours accessible to this compound. Alternatively, delivery of anticancer compounds into the brain can be achieved using Ommaya reservoirs. These medical devices allow intraventricular injection of the drugs, even in very young patients. 37 Development of resistance by cancer cells has proven to be one of the most frequent causes of targeted therapy failure and may account for the low efficacy of therapies targeting the Shh pathway in MB. 38,39 mTORC1 pharmacological inhibition can overcome the acquired resistance to Shh targeted therapy in Shh group MBs. 40 Our study shows that inhibition of LAT1 leads to a strong decrease in mTORC1 activity, suggesting that the use of JPH203 may be relevant to bypass resistance to Shh targeted therapies. We also show that despite an adaptive response via an up-regulation of EAA transporter expression, MB cells treated for several months with JPH203 never acquired the capacity to overcome the cytotoxic and cytostatic effects of this compound. This result suggests that MB cells may not have the ability to adapt to EAA import inhibition and subsequent mTORC1 inactivation. Even if encouraging, our results still need in vivo validation. In particular, the ability of JPH203 to pass the blood-brain barrier and its toxicity towards developing organisms still require thorough investigation.