Peer review of: Pancreatic cancers suppress negative feedback of glucose transport to reprogram chromatin (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
This study by Bechard et al builds on the authors’ previous findings that cells derived from distant metastases of pancreatic 
ductal adenocarcinoma (PDAC) are specifically dependent on the oxidative pentose phosphate pathway (ox-PPP) enzyme, 
phosphogluconate dehydrogenase (PGD), for growth in vitro. This work suggested that a distinct metabolic program 
supports metastatic spread and is linked to extensive changes in chromatin regulation. Since multiple studies show that 
primary PDAC and metastasis share the same genetic profiles, the exploration of the non-genetic basis of metastatic 
outgrowth and dependency is an important area of study and the role of altered metabolic pathways in the survival of 
distant metastatic PDACs is a new paradigm in metastasis research. In the present study the authors have examined the 
impact of PDG on the regulation of glucose uptake. They find that the presence of high levels of PDG in cells derived from 
distant metastasis generates a positive feedback pathway to maintain high glucose uptake. They report that thioredoxin-
interacting protein (TXNIP), an inhibitor of cellular glucose uptake, is reduced in these cells compared to cells from 
peritoneal metastasis, and that PGD knockdown increases TXNIP levels and suppresses glucose u ptake. They relate these 
effects to the inhibition of the MondoA transcription factor by accumulation of the PDG substrate, 6PG. They also show that 
TNXIP overexpression specifically reduces the growth of 3D organoids from distant metastasis. Finally, the authors show in 
patient tumor samples, that TXNIP staining is reduced distant metastases compared to peritoneal metastases. 
 
These studies are novel and of interest and the data showing the relationship between PDG, TXNIP and glucose uptake are 
clear-cut. The primary concern is that limitations in the model systems restrict the conclusions that can be drawn. The 
study is primarily framed around the difference between peritoneal and distant metastasis. Moreover, most experiments 
compare a single pair of peritoneal vs distant metastasis models. It is not clear whether the differences noted relate to 
features particular to the peritoneal metastasis used as a reference rather than properties acquired as primary tumors 
undergo distant metastatic spread. Also, the generality of the observations is not established. Finally, the cell lines used in 
this manuscript do not form tumors in xenograft assays, and so their relevance to the in vivo situation is uncertain. While 
it is understood that these models are difficult to obtain, some additional support would be needed to confirm the 
conclusions of the paper. 
 
1) Figure 1B should include additional primary tumor-derived lines in the comparison. The present analysis includes long-
established cell lines from repositories and a single peritoneal met sample as references. A more relevant and complete 
comparison would utilize matched early passage primary and mets (preferably) or a number of early passage primary PDA 
lines. 
2) The functional studies are based on a single pair of cell lines (38Per vs 38Lg). The phenotypes should be tested in 
multiple cell lines. 
3) Please include immunoblots for TXNIP (Fig 1D) and the histone marks (Fig 3a). 
4) The rationale for excluding primary tumors in the IHC analysis in Figure 4 is not clear. As noted above, it is not certain 
whether TXNIP is decreased in metastasis compared to primary PDAC or whether TXNIP levels are specifically elevated in 
peritoneal metastases. 
5) The regulation of nucl-cyto translocation of MondoA by 6PG is an important observation (Extended data 3). The authors 
should clarify how these metabolites are treated in this experiment. Since phosphorylated compounds like 6PG and G6P 
are poorly cell-permeable, it is important to show the intracellular concentration of the 3PG after exogenous addition. 
6) Are steady-state 6PG levels reduced in the PGD-high cells? Also, in 
7) The impact of the study would be enhanced by some insight into how inhibition of PGD regulates mondoA -TXNIP axis? 
8) In Figure 4 the authors used siRNA to knockdown TXNIP and performed 3D growth assays over 7-21 days. Since siRNA 
causes transient knockdown the authors may want to consider additional controls. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This is an interesting and well done manuscript that describes a role for altered metabolism in distant pancreatic cancer 
metastatic lesions. This manuscripts follows others from the same group and should be of high general interest. The data 
is convincing and clearly shows that glucose uptake is upregulate in distant lesions that are dependent on PGD and that 
high PDG levels result in suppression of TXNIP. This pathway dissection is followed up by a reasonable set of experiments 
to validate the biological significance of their working model. Particularly interesting is the finding that in PDG tumors the 
levels of H3K27ac are upregulated, perhaps suggesting a broad rewiring of gene expression in this subgroup of lesions. On 
the downside, the manuscript is largely descriptive and lacks mechanistic depth. This is a major concern. 
1. The proposal is that TXNIP loss leads to an increase in glucose uptake, which is consistent with a body of literature and 
clearly shown here. They propose that the increase in glucose uptake leads to an increase in Acetyl-CoA resulting in an 
ncrease levels of histone acetylation. which is not explicitly tested here. This is important given TXNIP’s many roles that 

seem to be unrelated to functions in glucose uptake and/or metabolism. For example, TXNIP loss in MEFs leads to 
activation of PI3-Kinase, which could easily account for their observations. 
2. The staining for H3K27Ac is convincing as is the ChIP profiling for H3K27Ac at CDH2 shown in Figure 3b. At least a few 
more TXNIP regulated targets shown in figure 3b, should be evaluated. It may be just as easy to perform a ChIP-seq 
experiment for H3K27ac. 
3. PGD loss increases TXNIP expression and the authors’ have some data that induction is MondoA-dependent. They should 
test this directly and determine whether TXNIP induction depends on MondoA as they data as it now stands is only 
correlative. It would also be valuable to determine if PGD loss leads to an increase in G6P as this is the primary regulator 
of MondoA transcriptional activity. Likewise measuring G6P levels in PGDhigh lesions would also be informative. 
Determining how G6P regulates MondoA activity is a big ask and is beyond the scope of this manuscript. 
4. This is more of a minor concern. The author’s present their data as consistent with both positive and negative feedback 
loops. Clearly this is complicated, but the description was cumbersome in my opinion and they don’t really test the 
regulatory loops rigorously. It may be more straight forward just to describe the metabolic wiring of cells in the PDGhigh 
and the PDGlow states. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Bechard et al study distant vs peritoneal metastasis in PDAC. They find that distant metastasis with high PGD (identified in 
a previous paper) have low expression of TXNIP. Reduced expression of TXNIP is known to lead to increased GLUT1 
expression and therefore glucose uptake. Accordingly, they find that PGD regulated TXNIP expression impacts on glucose 
uptake. In line, exogenous TXNIP decreased 3D growth of distant metastasis but not peritoneal metastasis (while TXNIP 
silencing impair peritoneal metastasis) and TXNIP was less expressed in distant metastasis from patients. The main finding 
of this paper is that PGD regulates (by an unknown mechanism) TXNIP expression and that this decreased expression is 
important for the previously described change in histone acetylation. 
 
Major comments: 
1) The focus on a switch from a negative to a positive feedback loop is confusing and not supported by data. In my 
opinion, the data simply suggest that PGD is a negative regulator of TXNIP. 
2) Accordingly, it is required that title and abstract reflect the actual data presented in the manuscript rather than a 
speculative interpretation of the data. 
3) The authors mention several times the context of evolution and non-genetic driver. These terms should be removed 
from the manuscript or experiments should be provided to support the claims. For example, is PGD overexpression or 
TXNIP deletion sufficient to drive a non-metastatic PDAC cell to lung or liver? 
4) Evidence should be provided that altering PGD or TXNIP prevents in vivo metastasis formation.