Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP’s substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

The authors analyzed a panel of 18 primary GBM for MTA and SAM levels and observe no significant difference in levels between MTAP intact and low MTAP expressing tumors, though MTA levels are generally higher in the lines with lower MTAP expression. They demonstrate that MTAP staining is intact in MTAP-WT and stromal tissue in human samples while absent in MTAP-deleted tumors. This is also true in xenografts. Having demonstrated that in vivo MTAP is present in the tissues surrounding the cell, the authors show by co-culture of MTAP-deleted and MTAP-intact cell lines that elevated MTA in the media of MTAP-deleted lines can be cleared from the media when MTAP-WT cells are present. As an indirect measure of PRTM5 activity (which is inhibited by elevated MTA), PRMT5 activity increases, leading to increased levels of SDMA, with co-culture leading to MTA clearance from the media.
Overall, this is an interesting study but there are a couple critical issues that need to be addressed to make the data more convincing. Specifically, the authors need to strengthen data surrounding the lack of correlation between MTAP and MTA in vivo. MTA elevations have previously been reported in MTAP-deleted tumors and I am not convinced by how they defined MTAP-deleted in their GBM sample population. Theoretically, MTAP-deleted tumors should exhibit no MTAP expression by western blot, and there are certainly patient-derived GBM glioma models in use that do not have detectable protein levels of MTAP. Here, however, the authors are using MTAP-deletion and low MTAP interchangeably but they have not provided sufficient evidence that this is appropriate. This makes me wonder if a significant correlation between MTAP loss and MTA elevation might be found if their definition/threshold for "low MTAP expression" was more stringent.
• MTAP-deletion and "low MTAP expression" are not necessarily the same and I worry that the authors assumption of this leads to inaccurate conclusions. How exactly was MTAP expression measured in Supplementary Figure 2 and what is the label of the y-axis. Is this protein level (western blot/IHC), mRNA, something else? mRNA level is not always equivalent to protein abundance. A tumor with homozygous deletion of 9p21 would be CN = 0 and therefore no expression at all. • Along these lines, MTAP expression is observed in the tumors that the authors are calling "MTAP deleted". Low (but present) MTAP protein level may be sufficient to regulate MTA levels. In Figure 3, it would be helpful to see how MTA levels correlate with amount of MTAP protein present by western blot.
• Data in Supplementary Figure 4 do not support your other data -these show that MTA levels (normalized to mean or normalized to SAM) are significantly higher in MTAP-deleted tumors. Why the discrepancy? And, are these tumor truly MTAP-deleted or MTAP-low? • Supplementary Figure 6D and it's conclusions are based on assumptions and not known genotypes of the patients being sampled. This is a weak argument. • Figure 1A is not data but summary of the pathway -perhaps the TCGA data should be omitted since this is not primary data. • It is difficult to read the y-axis units on Figure 2C -is this 10Z? • Please be cautious in the discussion of MAT2A as a target. Though MAT2A's important cellular role is noted, many targeted therapies are aimed at essential genes (ex EGFR null mice have early lethality) -it is the therapeutic window that is attempted to be exploited with MAT2A inhibition in the setting of MTAP deletion. • The GBM tumors label is out of place on Suppl. Fig 3. Reviewer #3 (Remarks to the Author); expert on MTAP-deleted targeting: A major conclusion from this paper is that MTA Is excreted by GMB cells and is metabolized by stromal cells. Evidence for products of metabolism of MTAP would further strengthen their conclusions,

Reviewer Comments:
Reviewer #1 (Remarks to the Author); expert on metabolism: In the paper entitled "Metabolomic profilings do not corroborate elevation of Methylthioadenosine in MTAP-deleted Glioblastoma" Barekatain and colleagues investigate the metabolic changes in cells deficient for the enzyme Methylthioadenosine Phosphorylase (MTAP), lost in a subset of glioblastoma tumours and cell lines, in conmbination with the tumour suppressor P16. They observe that, contrary to the expectations, MTAP-deficient cells exhibit only a modest intracellular accumulation of methylthioadenosine (MTA, the substrate of MTAP), and this metabolite is mostly released in the extracellular milieu. In addition, they propose that secreted MTA can be taken up by stromal immune cells, limiting the overall accumulation of this metabolite in situ. These findings call for caution in inferring metabolic behaviour from tissue culture experiments, and for therapeutic intervention of MTAP-deficient tumours based on the expected accumulation of MTA. Although these results are relevant some concerns should be addressed before publication.
1) The authors propose that MTAP-deficient cells do not accumulate MTA in vivo is because its accumulation is a "predominantly extracellular phenomenon" and that stromal cells metabolize the secreted MTA. Yet, recent work (Sanderson et al (Science Advances 2019), has highlighted the lack of correlation between MTAP status and MTA accumulation and ascribed it to nutrient availability and heterogeneity in one-carbon metabolism. Regrettably, the authors discuss this important work only briefly at the end of the discussion. It is recommended that the authors introduce the work of Sanderson early on, given its relevance, and present the data in the context of these earlier findings. In addition, they could use the data shown in this paper to further validate their hypothesis and their analyses of external datasets. Finally, and more importantly, the authors should assess whether the accumulation and secretion of MTA in cells are confirmed using a more physiological medium (Plasmax or HPLM), where the concentration of metabolites that affect methionine pathway reflects those found in the blood. Indeed, it is possible that the lack of accumulation of MTA in MTAP-deficient tumours is due to the depletion of specific nutrients, including methionine and cysteine, in vivo, rather than by secretion and scavenging. Using a physiological medium would allow a more faithful comparison between their observation in vitro and vivo, providing strength to the central hypothesis.
 In response to point A: The reviewer's comment is spot on-we should indeed have discussed the seminal paper by Sanderson et al. 1 Figure). At the same time, MTA levels positively correlate not just with methionine, but with most other amino acids (including serine) and the sum of all amino acids, with higher statistical significance than methionine. This suggests that whatever association between methionine and MTA is unlikely to be specific and causative and more likely reflects the cellularity of individual tumors.   Figure 1F), it could be possible that the difference in MTA levels between MTAP-deleted and WT cells cultured in restricted cysteine conditions became more significant if cells were grown for a longer time (i.e., 24 hours). As mentioned in our paper, we do not explicitly rule out the possibility that nutrient differences cause the discrepant observation of lack of MTA accumulation in primary human tumors with MTAP deletions. mass-spect cores), we did not observe any significant difference (increase or decrease) in levels of methionine as well as cysteine and serine between MTAP-deleted and intact human GBM tumors. This indicates that the variation between methionine and cysteine levels between MTAP-deleted and intact GBM tumors is minimal. Moreover, to see if the levels of methionine and cysteine in GBM tumors are extremely lower than brain, we compared the levels of these metabolites in a mouse brain (as an illustrative of a human brain since we do not have any normal human brains for such comparisons) with primary GBM tumors from the same dataset (The Below Figure, Panel A). We did not observe methionine and cysteine levels to be significantly lower in GBM than in the normal brain. (nanomoles) found in the cell pellets and paired conditioned media ( Figure 1H). For these experiments, the entire cell pellet was extracted; an aliquot of media was extracted, the concentration was determined using deuterium-labeled MTA as an internal standard. The concentration of MTA in conditioned media was corrected for the percentage of media to extraction buffer. The absolute amounts of MTA in cell pellet and media were determined by multiplying the concentration by the total volume of cell and media extract.
The key points from these comparisons are: I. Conditioned media has dramatically higher absolute amounts of MTA in MTAPdeleted (24.637  5.357 nanomoles, meanSD) vs. MTAP-intact cells (0.0060.003 nanomoles, mean SD). This can also be seen as ion count levels in Figure 1E.   proportion of GBM mass and express WT levels of MTAP, there is no expectation that a western blot performed on a lysate of the bulk resected tumor (containing an admixture of malignant MTAP-null cancer cells and MTAP-expressing stromal cells) would show a complete absence of MTAP.
 On average, the MTAP-deleted tumors express a lower amount of MTAP protein levels by the western blot than MTAP-intact (below graph). However, some expression of MTAP deriving from MTAP-WT stromal cells will always remain. This is why immunohistochemistry is necessary and allows clear discrimination of the residual expression of MTAP in non-transformed stromal cells versus its absence in glioma cells. To illustrate that stroma exists in bulk resected tumors and lysates derived from it, we immunoblotted myeloid marker (IBA1, as microglia/macrophages constitute the largest tumor stroma component in GBM, Figure 2G, and Supplementary Figure S5). All tumors show some levels of IBA1 expression, which in MTAP-  The presence of microglia in a tumor is confirmed by the microglial markers IBA1 in tumor lysates but not seen in cells in culture.
The non-zero levels of MTAP protein for the homozygous MTAP-deleted HF3174 tumor (marked with red arrow) is more evident in higher exposed band of MTAP. In contrast, no MTAP expression is detected in the lysate of MTAP-deleted cells (U87).

Figure 2H
deleted tumors, nicely correlates with residual MTAP. Interestingly, the reviewer mentioned that the MTAP-deleted HF3174 tumor (a red arrow in Figure 2G) as the "only one MTAP-deficient tumour shows genuine loss of MTAP protein levels." Note the lower levels of IBA1 protein (low myeloid stroma infiltration) for this tumor (HF3174), resulting in low but non-zero expression of MTAP compared to other homozygous MTAP-deleted tumors. Public Domain (TCGA data)

Supplementary Figure S16D
 Moreover, the reviewer had concerns regarding how we identified and classified homozygous MTAP-deleted tumors in our study. Homozygous MTAP-deleted and intact GBM tumors in Figure  2 (HF series) were determined by array cGH (array comparative genomic hybridization) in the studies described by Kim et al. 7 and Wang et al. 8 In sum, DNA was isolated from whole tumors and hybridized with SNP 6.0 affix arrays. Copy numbers were corrected for tumor cellularity and whole-genome duplications. The MTAP deletion data ( Figure 2B) was obtained from the publicly deposited data from Kim et al. 7 We further confirmed MTAP-status of tumors by immunohistochemistry against MTAP using a validated antibody (as described in the manuscript   In the revised manuscript, the methionine salvage representation is placed in Figure 1A. Moreover, the proposed mechanism of regulation of PRMT5 by MTA moved to the supplementary information (Supplementary Figure S1).

Reviewer #2 (Remarks to the Author); expert on GBM:
Barekatain and colleagues perform analysis of previously published and new metabolic profiling studies to suggest that excess MTA in MTAP-deleted gliomas is secreted into the extracellular environment and processed by MTAP-intact cells within the tumor microenvironment, leading them to propose that MTAP-deletion/excess MTA may not be a good therapeutic target. Homozygous deletion of 9p21 is frequent in GBM, leading in deletion of well-known tumor suppressor CDKN2A and collateral deletion of MTAP, which has provided prior motivation for targeting this vulnerability.
MTAP has a role in adenine and methionine salvage pathways and deletion of MTAP leads to elevated MTA in many in vitro models; there are now strategies under investigation that exploit the elevation in MTA (PRMT5i and MAT2Ai). The authors note that despite much in vitro data, there is currently no evidence to support that MTA levels are elevated in MTAP deleted tumors, such as primary GBM. Further, upon analysis of published metabolomics data, they observe that MTA elevation is primarily extracellular and there are no tumors with extreme MTA elevation. The authors analyzed a panel of 18 primary GBM for MTA and SAM levels and observe no significant difference in levels between MTAP intact and low MTAP expressing tumors, though MTA levels are generally higher in the lines with lower MTAP expression. They demonstrate that MTAP staining is intact in MTAP-WT and stromal tissue in human samples while absent in MTAP-deleted tumors. This is also true in xenografts. Having demonstrated that in vivo MTAP is present in the tissues surrounding the cell, the authors show by co-culture of MTAP-deleted and MTAP-intact cell lines that elevated MTA in the media of MTAP-deleted lines can be cleared from the media when MTAP-WT cells are present. As an indirect measure of PRTM5 activity (which is inhibited by elevated MTA), PRMT5 activity increases, leading to increased levels of SDMA, with co-culture leading to MTA clearance from the media. Overall, this is an interesting study but there are a couple critical issues that need to be addressed to make the data more convincing. Specifically, the authors need to strengthen data surrounding the lack of correlation between MTAP and MTA in vivo. MTA elevations have previously been reported in MTAPdeleted tumors and I am not convinced by how they defined MTAP-deleted in their GBM sample population.  Another study regarding measuring MTA levels in human tumors done by Stevens et al. 18 in which authors measured MTA levels in a small number of primary human melanoma tumors (n=5). However, Stevens et al. 18 did not attempt to determine whether MTA levels were higher in MTAPdeleted versus MTAP-intact primary melanoma tumors. This clarification is critical given that MTAP homozygous deletion only occurs in ~18% of melanomas, and the sample size is very small (n=5).
 We should emphasize that our conclusion of "no significant elevation of MTA in homozygous MTAP-deleted human GBM tumors" (data are shown in Figure 2 and Supplementary Figure S5) is based on tumors with verified homozygous MTAP deletion. The MTAP status of these tumors was determined from previous cGH studies (CNV data in Wang et al. 8 (Figure 2G and Supplementary Figure S5). All tumors show some levels of IBA1, verifying myeloid stromal infiltration. In Figure  2G, note the homozygous MTAP-deleted HF3174 tumor has the lowest IBA1 compared to other homozygous MTAP-deleted tumors (indicating least stroma-containing tumor of this dataset), which also expresses the lowest but non-zero levels of MTAP (red arrows in Figure 2G).  The non-zero levels of MTAP protein for homozygous MTAP-deleted HF3174 tumor (marked with red arrow) is more evident in the higher exposed band of MTAP. In contrast, no MTAP expression is detected in the lysate of MTAP-deleted cells (U87).  To further validate that IBA1-positive myeloid cells drive the non-zero MTAP expression in bulk MTAP-deleted GBM tumors, we investigate the correlation between MTAP mRNA and AIF1 (the official gene symbol of IBA1, microglia/macrophages marker) mRNA levels among GBM tumors in an independent data set (TCGA 6 ). Supplementary Figure S16D shows that there is a significant positive correlation between mRNA levels of MTAP and AIF1 in MTAP-deleted tumors vs. intact ones (but no positive correlation between MTAP and AIF in MTAP-intact tumors, fully consistent with the explanation, as in MTAP-intact tumors, both glioma and stromal cells express MTAP). This figure also indicates that homozygous MTAP-deleted GBM tumors, on average, have lower but non-zero levels of MTAP mRNA levels compared to MTAP-intact tumors, supporting our western blot data in Figure 2G. strong MTAP staining in the normal mouse brain (Supplementary Figure S12-S14), confirming negligible stromal infiltration in human xenografted tumors.

Figure 2H
Here, however, the authors are using MTAP-deletion and low MTAP interchangeably but they have not provided sufficient evidence that this is appropriate. This makes me wonder if a significant correlation between MTAP loss and MTA elevation might be found if their definition/threshold for "low MTAP expression" was more stringent. MTAP-deletion and "low MTAP expression" are not necessarily the same and I worry that the authors assumption of this leads to inaccurate conclusions.
 First, please note that we did not use the "MTAP-deletion" and "low MTAP expression" interchangeably. Our conclusion of "insignificant elevation of MTA in homozygous MTAPdeleted GBM tumors" has derived from comparing MTA levels between homozygous MTAPdeleted tumors and MTAP-intact, as defined by genomic array cGH studies performed in reference 7   In the first submission of the paper, we first discussed MTA levels in GBM tumors from public domain data, and then we discussed our metabolomic profiling data. For the public domain data (Previously Supplementary Figure S2 and now Supplementary Figure S6), we do not have information on MTAP deletion status, and we do not claim to know MTAP status for each tumor.
In the new version of the paper, we first discuss our profiling data (MTA levels in GBM tumors with verified MTAP-deletion status, Figure 2 and Supplementary Figure S5). We then used the public domain data to support our argument as independent measurements. We hope changing the flow of the paper resolves this confusion. Please see the reply to the following comment regarding clarification of previously Supplementary Figure S2 and now Supplementary Figure S6.
 The MTAP-deletion status of GBM tumors (HF series) in Figure 2 was determined by array comparative genomic hybridization (array cGH) studies 7,8 and further verified by immunohistochemistry. The genomic copy number variation of HF series tumors ( Figure 2B) was profiled previously 7 , and the MTAP deletion data was obtained from this publication's publicly deposited data. Briefly, DNA was isolated from whole tumors and hybridized with SNP 6.0 affi arrays. Copy numbers were corrected for tumor cellularity and wholegenome duplications. Our assignment of homozygous MTAP deletion versus MTAP-WT status for the HF series' tumors agrees with the CDKN2A status of the tumors assigned by experts in the genomic field in Kim et al. 7 paper. Please How exactly was MTAP expression measured in Supplementary Figure 2 and what is the label of the y-axis. Is this protein level (western blot/IHC), mRNA, something else? mRNA level is not always equivalent to protein abundance. A tumor with homozygous deletion of 9p21 would be CN = 0 and therefore no expression at all. Along these lines, MTAP expression is observed in the tumors that the authors are calling "MTAP deleted". Low (but present) MTAP protein level may be sufficient to regulate MTA.
 This point goes to the same principle elaborated on in the previous reply: When assayed at the bulk tumor level, MTAP-deleted tumors still express MTAP because of non-malignant, MTAPexpressing stromal cells. So, we agree with the reviewer that on its own, when measured in bulk tumors, low MTAP expression does not prove homozygous MTAP-deletion, yet, MTAP-deleted tumors, as defined by genomic copy number, show on average, lower expression of MTAP even when assayed in bulk; this is evident both in our western blot data as well as in RNA seq data from TCGA 14 (Supplementary Figure S6G). For data shown in the initial manuscript in Supplementary Figure S2 (public domain MTAP mRNA with MTA metabolite levels, Prabhu et al. 19 ) and now in the revision shown in Supplementary Figure S6A- Figure S6G). Note that the vast majority (though clearly, not all) of the MTAP-genomically deleted (blue dots) tumors fall in first and second quartiles of low MTAP expression, while the bulk of MTAP-intact tumors fall into the third and fourth quartiles of higher mRNA MTAP expression. Based on this, it is reasonable to infer that in the Supplementary Figure S6A-F (Prabhu et al. 19 ), if tumors are ranked by quartiles of MTAP expression, the vast majority of MTAP-deleted cases will be found in first and second quartiles. Data on Supplementary Figure S6 are provided as an additional example to support our conclusion from tumors with known MTAP-deletion status (Figure 2 and Supplementary Figure S5).
In Figure 3, it would be helpful to see how MTA levels correlate with amount of MTAP protein present by western blot.
 Pleases see the below graph, which shows the MTA levels versus the amount of protein levels present by Western blot. No significant correlation was found.
Data in Supplementary Figure 4 do not support your other datathese show that MTA levels (normalized to mean or normalized to SAM) are significantly higher in MTAP-deleted tumors. Why the discrepancy? And, are these tumor truly MTAP-deleted or MTAP-low?
 Please note that Supplementary Figure S4 illustrates MTA and SAM levels in cells in culture and not human GBM tumors. We re-wrote the figure legend to emphasize that these data represent metabolites levels of cells in culture, not human tumors. We are all in agreement, for cells in culture without stromal cells, MTA levels are higher in MTAP-deleted cancer cell lines than WT. These data, which were re-plotted from the supplementary information of different published studies 9,20 , indicate that SAM levels are not different between MTAP-deleted and wild-type cancer cell lines in culture. These data justify using SAM as an intracellular metabolite for the normalization of MTA.
Supplementary Figure 6D and it's conclusions are based on assumptions and not known genotypes of the patients being sampled. This is a weak argument.
 Supplementary Figure S6 is now shown as Supplementary Figure S11. We agreed that for this figure, knowing the MTAP deletion status of tumors will strengthen the argument. Unfortunately, such data were not collected and are not available. However, we want to point out that the argument is based on comparing a group with a high incidence of homozygous MTAP-deletion (grade IV GBM ~50% MTAP-deleted) with lower or zero incidence of homozygous MTAP-deletion (lowgrade glioma and normal brain). Homozygous MTAP deletion frequency can reach 50% 6 in GBM tumors, while its incidence is about 10% 20 in low-grade glioma and zero in a normal brain. Using the binomial distribution, the chance of having at least one MTAP-deleted tumor (among 10 tumors) for Supplementary Figure S11A is 99.9%. The probability of having at least one MTAPdeleted tumor-among 6 tumors-for Supplementary Figure S11C-S11E is 98.4%. However, the probability of having MTAP-deleted samples for the normal brain (Supplementary Figure S11A) and low-grade glioma (Supplementary Figure S11C-S11E) is zero and 46%, respectively. In our argument in the manuscript, we clearly state that the argument is based on likelihood (assumption) and not knowing the patients' genotypes.
MTA levels vs. MTAP protein levels from western blot for the HF series tumors in Figure 2. No significant correlation was found between MTAP protein levels vs. MTA levels. Figure 1A is not data but summary of the pathwayperhaps the TCGA data should be omitted since this is not primary data.
 We moved TCGA data to Supplementary Figure S1. TCGA data are public domain data free to use in publications and critical to supporting genomic studies.
It is difficult to read the y-axis units on Figure 2C is this 10Z?
 Please note that the y-axis on this figure is 10 7 . We re-plotted the figure to make the y-axis to be more apparent.
Please be cautious in the discussion of MAT2A as a target. Though MAT2A's important cellular role is noted, many targeted therapies are aimed at essential genes (ex EGFR null mice have early lethality)it is the therapeutic window that is attempted to be exploited with MAT2A inhibition in the setting of MTAP deletion. For mechanisim releted to other inhibiotrs it may prove useful to others.
 We fully agree with the reviewer that MAT2A inhibition may very well prove efficacious for cancer treatment, as this is an essential gene. However, our argument centers on whether a therapeutic window for MAT2A and PRMT5 inhibitors generated by homozygous MTAP-deletion is translatable to human primary GBM tumors. To address the reviewer's comment and to clearly state that our results do not detract from other avenues of utilizing MAT2A inhibitors in cancer treatment, we have modified our discussion as below: "That MTAP deletions can be leveraged as a point of selective vulnerability in various cancers has spurred much excitement. Our analysis challenges whether the metabolic conditions required for therapies to exploit vulnerabilities associated with elevated MTA/ MTAP-deletions are present in primary human tumors, giving pause to whether these would translate to the clinic. Yet, our findings do not rule out that MAT2A and or PRMT5 inhibitors could prove useful in oncology. Merely, such inhibitors would have to achieve a therapeutic window through a mechanism other than MTA accumulation. Indeed, recent research has already pointed towards MTAP-deletion independent sensitization mechanism to PRMT5 inhibition 47 . Some GBM tumors in our dataset showed quite low levels of SDMA levels -suggesting low PRMT5 activity-without MTAPdeletions (Figure 2). Perhaps such tumors with low PRMT5 activity could be suitable candidates for targeted therapies against PRMT5 or MAT2A, provided a reliable molecular marker can be found to identify them." The GBM tumors label is out of place on Suppl. Reviewer #3 (Remarks to the Author); expert on MTAP-deleted targeting: A major conclusion from this paper is that MTA Is excreted by GMB cells and is metabolized by stromal cells. Evidence for products of metabolism of MTAP would further strengthen their conclusions.
 To address the concerns raised by the reviewer, we performed a series of experiments to 1) determine if the release of functional MTAP enzyme by MTAP-WT cells into the media is responsible for eliminating exogenous MTA from conditioned media and 2) isotope labeling studies to demonstrate that exogenous MTA is taken up by MTAP-intact cells and converted to methionine, via MTAP and the methionine salvage pathway.  Figure 4D shows the deuterium's fate from the labeled D3-MTA into methionine (methionine salvage pathway) and SAM (polyamine biosynthesis). This result indicates that extracellular MTA can be taken up and metabolized by MTAP-WT cells.