Cell circuits between leukemic cells and mesenchymal stem cells block lymphopoiesis by activating lymphotoxin beta receptor signaling
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eLife assessment
This study describes a previously unrecognized positive feedback loop between leukemic cells and stromal cells impeding normal hematopoiesis mediated by lymphotoxin produced by cancer cells and its receptor expressed in stromal cells. These valuable findings will guide future research in both basic and clinical medicine. However, additional experimental evidence including more comparator groups would have further substantiated the authors' conclusions.
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Abstract
Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Using mouse models of ALL and AML, here we show that leukemic cells turn off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin α1β2 and activate lymphotoxin beta receptor (LTβR) signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin α1β2 expression in leukemic cells. Genetic or pharmacological disruption of LTβR signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage.
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Author Response
Reviewer #2 (Public Review):
In the current manuscript, Feng et al. investigate the mechanisms used by acute leukemia to get an advantage for the access to the hematopoietic niches at the expense of normal hematopoietic cells. They propose that B-ALLs hijack the niche by inducing the downmodulation of IL7 and CXCL12 by stimulating LepR+ MSCs through LTab/LTbR signaling. In order to prove the importance of LTab expression in B-ALL growth, they block LTab/LTbR signaling either through ligand/receptor inactivation or by using a LTbR-Ig decoy. They also show that CXCL12 and the DNA damage response induce LTab expression by B-ALL. They finally propose that similar mechanisms also favor the growth of acute myeloid leukemia.
Although the proposed mechanism is of particular interest, further experiments and controls are …
Author Response
Reviewer #2 (Public Review):
In the current manuscript, Feng et al. investigate the mechanisms used by acute leukemia to get an advantage for the access to the hematopoietic niches at the expense of normal hematopoietic cells. They propose that B-ALLs hijack the niche by inducing the downmodulation of IL7 and CXCL12 by stimulating LepR+ MSCs through LTab/LTbR signaling. In order to prove the importance of LTab expression in B-ALL growth, they block LTab/LTbR signaling either through ligand/receptor inactivation or by using a LTbR-Ig decoy. They also show that CXCL12 and the DNA damage response induce LTab expression by B-ALL. They finally propose that similar mechanisms also favor the growth of acute myeloid leukemia.
Although the proposed mechanism is of particular interest, further experiments and controls are needed to strongly support the conclusions.
1/ Globally, statistics have to be revised. The authors have to include a "statistical analysis" section in the Material and Methods to explain how they proceeded and specify for each panel in the figure legend which tests they used according to the general rules of statistics.
We apologize for the lack of details. This has been corrected in the revised manuscript.
2/ The setup of each experiment is confusing and needs to be detailed. Cell numbers are not coherent from one experiment to the other. As an example, there are discrepancies between Fig1 and Fig2. Based on the setup of the experiment in Fig.2 (Injection of B-ALL to mice followed by 2 injections of treatment every 5 days), mice have probably been sacrificed 12-14 days post leukemic cell injection. However, according to Fig.1, B cells and erythroid cells at this time point should be decreased >10 times while they are only decreased 2-4 times in Fig.2. This is also the case in Fig.4B-J or Fig.5D with even a lower decrease in B cells and erythroid cells despite a high number of leukemic cells. Please explain and give the end point for each experiment in each figure (main and supplemental).
We understand the reviewer concern but we’d like point out the following: kinetic experiments such as these were reproduced multiple times in the laboratory. However, when comparing side-by-side experiments performed over the course of several months discrepancies in the exact days when leukemia shuts-down hematopoiesis are bound to happen. This is because there are numerous variables at play that we can minimize to the extent possible, but we cannot completely eliminate. For example, we took all possible steps to work with stable batches of preB-ALL cells. However, it is impossible to be absolutely certain that the batch in one experiment is identical to another experiment. Cells have to be expanded for adoptive transfer, which inevitably carries some variability (all biological systems undergo random mutations, including purchased C57Bl6/J from reputable vendors); slight differences in ALL engraftment (i.e. injection variability) can occur such that kinetics may change by a couple of days, etc. The findings we reported here are highly reproducible: ALL shuts down lymphopoiesis and erythropoiesis acutely, less so myelopoiesis; that LTbR signaling is the major mechanism shutting down lymphopoiesis but not erythropoiesis; that ALLs up-regulate LTbR ligands when compared to non-leukemic cells of the same lineage and at a similar developmental stage; that CXCR4 and DSB pathways both promote lymphotoxin a1b2 expression. The exact kinetics of these experiments will vary, or at least carry a margin of error that is to the best of our capability impossible to eliminate.
3/ To formally prove that the observed effect is really due to LTab/LTbR signaling, the authors must perform further control experiments. LTbR signaling is better known for its positive role on lymphocyte migration. They cannot rule out by blocking LTbR signaling, that they inhibit homing of leukemic cells into the bone marrow through a systemic/peripheral effect, more than through an impaired crosstalk with BM LepR+ cells. They must confirm for inhibited/deficient LTbR signaling conditions, as compared to control, that similar B-ALL numbers home to the BM parenchyma at an early time point after injection. Furthermore, they cannot exclude that the effect on the expression of IL7 (and other genes), and consequently the effect on B cell numbers, is not simply due to the tumor burden. Indeed, B-ALL numbers/frequencies are different between control and inhibited/deficient signaling conditions at the time of analysis. The analyses should thus be performed at similar low and high tumor burden in the BM for both control and inhibited/deficient LTbR signaling conditions.
We performed ALL homing experiments into control and LTbR∆ and found no significant differences in ALL frequency or number in BM 24h after transplantation. These data have been included in Figure 4A.
We also performed experiments to control for the number of ALL cells in the bone marrow. Briefly, we compared the impact of 3 million WT ALLs with that of 3 and 9 million Ltb-deficient ALLs on Il7-GFP expression in BM MSCs. The number of Ltb-deficient ALLs in the BM of mice recipient of 9 million ALLs was equivalent to that of mice that received 3 million WT ALLs 7 days after transplantation. Importantly, Il7 was only downregulated in mice transplanted with WT ALLs. These data have been included in Figure 4R and 4S.
4/ LT/LTbR signaling is particularly known for its capacity to stimulate Cxcl12 expression. How do the authors explain that they see the opposite?
The reviewer is alluding to a well-known role of LTbR signaling as an organizer of immune cells in secondary lymphoid organs such as spleen and lymph nodes, and particularly its role in promoting CXCL13, CCL19, CCL21 production by fibroblastic reticular cells of these organs. Both the B cell follicle and the T-zone do not express CXCL12 abundantly. Furthermore, in the B cell follicle niche, LTbR signaling is critical for the maturation of Follicular Dendritic Cells, yet FDCs hardly produce CXCL12 as well. So, while LTbR is a well-known regulator of cell organization through the production of homeostatic chemokines and lipid chemoattractants, CXCL12 itself is not one of the major chemokines controlled by this pathway. In summary, we do not think our data is in any way incompatible with prior studies on the LTbR pathway, and even if it was, to our knowledge this is the first study on cell-intrinsic effects of LTbR signaling in BM MSCs.
5/ The authors show that CXCL12 stimulates LTa expression in their cell line. They then propose that CXCR4 signaling in leukemic cells potentiates ALL lethality by showing that a CXCR4 antagonist reverses the decrease in IL7 and improves survival of the mice. This experiment is difficult to interpret. CXCL12 has been shown to be important for migration/retention of B-ALL in the BM and the decreased tumor burden is probably linked to a decreased migration more than an impaired crosstalk with LepR+ cells (see also point 3). If CXCL12 increases LTab expression, CXCR4 blockade should do the opposite. This result should be presented. The contradiction is that if B-ALLs induce a decrease in CXCL12 in the BM (in addition to IL7) and that CXCL12 regulates LTab levels, leukemic cells should be exhausted. Similarly, IL7 has been previously shown to stimulate LTab expression and B-ALL cells express the IL7R. Again, a decrease in IL7 should be unfavorable to B-ALL. How do they explain these discrepancies?
We thank the reviewer suggestion of testing the impact of CXCR4 blocking in vivo on LTa1b2 expression. We performed these experiments which have now been included in the revised manuscript (Fig. 5C and 5D). In summary, we observed reduced LTa1b2 on ALLs transplanted into mice treated with AMD3100, a well-known CXCR4 antagonist. These data also show that CXCR4 signaling is not the only mechanism driving LTa1b2. These results further strengthen the main conclusions of the manuscript. Finally, to our knowledge no study has reported Lymphotoxin a1b2 upregulation in B-ALLs by IL-7.
6/ In Supp 4A, RAG-/- mice are blocked at the pro-B cell stage and do not have pre-B cells. Please compare LTa and LTb expression by Artemis deficient pre-B cell to wt pre-B cells. In this experiment, the authors show that similarly to B-ALL artemis-/- pre-leukemic pre-B cells express high levels of LTab and induce IL7 downmodulation. Using mice deficient for LTbR in LepR+ cells, they show that IL7 expression is increased. However, in opposition to leukemic cells (see Figure 4F), pre-leukemic cells are increased in absence of LTab/LTbR signaling. Please explain this discrepancy. The authors use only one B-ALL model cell line for their demonstration (BCR-ABL expressing B-ALL). Another model should be used to confirm whether LTab/LTbR signaling does favor leukemic/pre-leukemic B cell growth.
We apologize for the confusion. The mice that were used in this study were initially described by Barry Sleckman and colleagues (Bredemeyer et al. Nature 2008). Briefly, they crossed Artemis-deficient mice with VH147 IgH transgenic and EμBcl-2 transgenic mice to generate mice in which B cell development is arrested at the preB cell stage. The Vh147 heavy chain allows their development to the pre-BCR+ preB cell stage but Artemis deficiency prevents Rag protein re-expression and hence B cell can’t recombine light chain genes. The EμBcl-2 transgene allows preB cells to survive despite carrying unrepaired double-strand DNA breaks (DSB).
Regarding the discrepancy noted by the reviewer we argue that this is not a discrepancy. While ALLs can grow in vitro and in vivo in the absence of IL7, non-leukemic developing B cells are strictly IL7 dependent. PreB cells carrying unrepaired DSBs still express IL7 receptor and although no data is currently available on whether these cells are also IL7-dependent, we speculate that they are. Because up-regulation of Lymphotoxin a1b2 in preB cells carrying unrepaired DSBs promotes IL7 downregulation we speculate that this mechanism may contribute to the efficient elimination of pre-leukemic preB cells in vivo. We revised the manuscript to include this explanation of the mouse model and discussion on how we think the LTbR pathway may play a role in pre-leukemic states.
Finally, the data presented in this study includes two distinct leukemia mouse models. It also includes data from human B-ALL and AML samples that is in agreement with the mouse data presented here. We respectfully disagree with the reviewer that a third model is needed to confirm a role for the LTa1b2/LTbR pathway in leukemia.
7/ Pre-B cells are composed of large pre-B cells (pre-BCR+) and small pre-B cells (pre-BCR-). BCR-ABL B-ALL cells express the pre-BCR. What is the level of expression of LTa and LTb by each of these 2 subsets as compared to BCR-ABL B-ALL?
This is a misconception. The difference between large and small preB cells is simply that large preB cells are in S/G2 phase of the cell cycle. Their increased size is a mere consequence of doubling DNA, protein, membrane content, etc.
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eLife assessment
This study describes a previously unrecognized positive feedback loop between leukemic cells and stromal cells impeding normal hematopoiesis mediated by lymphotoxin produced by cancer cells and its receptor expressed in stromal cells. These valuable findings will guide future research in both basic and clinical medicine. However, additional experimental evidence including more comparator groups would have further substantiated the authors' conclusions.
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Reviewer #1 (Public Review):
Leukemic cells are known to remodel bone marrow niche to promote their expansion and to suppress normal hematopoiesis. However, molecular mechanisms remain largely unknown. In this manuscript, authors developed new experimental models in mice to address this issue, using mouse BCR-ABL-driven ALL cells marked with YFP, or DOX-inducible MLL-AF9 AML cells. After transplantation of either of these cells, authors discovered suppression of host hematopoiesis. Using these systems, authors tested their hypothesis on lymphotoxin receptor-mediated interaction of the leukemic cells and stroma cells.
The main conclusions here are: 1) lymphotoxin signaling through its receptor mediates IL7 down regulation and alters gene expression related to inflammation etc. in stroma cells, 2) IL7 down regulation leads to reduction in …
Reviewer #1 (Public Review):
Leukemic cells are known to remodel bone marrow niche to promote their expansion and to suppress normal hematopoiesis. However, molecular mechanisms remain largely unknown. In this manuscript, authors developed new experimental models in mice to address this issue, using mouse BCR-ABL-driven ALL cells marked with YFP, or DOX-inducible MLL-AF9 AML cells. After transplantation of either of these cells, authors discovered suppression of host hematopoiesis. Using these systems, authors tested their hypothesis on lymphotoxin receptor-mediated interaction of the leukemic cells and stroma cells.
The main conclusions here are: 1) lymphotoxin signaling through its receptor mediates IL7 down regulation and alters gene expression related to inflammation etc. in stroma cells, 2) IL7 down regulation leads to reduction in B lymphoid cells but not myeloid cells, 3) lymphotoxin expression in leukemic cells is induced by DNA damage response, and 4) CXCR4, which is known to be induced in B cells in response to stroma cells, collaborates with DNA damage in induction of lymphotoxin in leukemic cells. Taken together, authors suggest that a positive feedback loop of leukemic cells and stroma cells for leukemic cell proliferation and normal hematopoietic suppression, involving lymphotoxin and CXCR4 in leukemic cells and lymphotoxin receptor in stroma cells. Generally, these conclusions and the model of the positive feedback regulation are supported, to a reasonable level, by the experimental results provided in the manuscript. However, some of the results show small effects of manipulations, leaving the pathological significance of the feedback model as a future issue.
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Reviewer #2 (Public Review):
In the current manuscript, Feng et al. investigate the mechanisms used by acute leukemia to get an advantage for the access to the hematopoietic niches at the expense of normal hematopoietic cells. They propose that B-ALLs hijack the niche by inducing the downmodulation of IL7 and CXCL12 by stimulating LepR+ MSCs through LTab/LTbR signaling. In order to prove the importance of LTab expression in B-ALL growth, they block LTab/LTbR signaling either through ligand/receptor inactivation or by using a LTbR-Ig decoy. They also show that CXCL12 and the DNA damage response induce LTab expression by B-ALL. They finally propose that similar mechanisms also favor the growth of acute myeloid leukemia.
Although the proposed mechanism is of particular interest, further experiments and controls are needed to strongly …
Reviewer #2 (Public Review):
In the current manuscript, Feng et al. investigate the mechanisms used by acute leukemia to get an advantage for the access to the hematopoietic niches at the expense of normal hematopoietic cells. They propose that B-ALLs hijack the niche by inducing the downmodulation of IL7 and CXCL12 by stimulating LepR+ MSCs through LTab/LTbR signaling. In order to prove the importance of LTab expression in B-ALL growth, they block LTab/LTbR signaling either through ligand/receptor inactivation or by using a LTbR-Ig decoy. They also show that CXCL12 and the DNA damage response induce LTab expression by B-ALL. They finally propose that similar mechanisms also favor the growth of acute myeloid leukemia.
Although the proposed mechanism is of particular interest, further experiments and controls are needed to strongly support the conclusions.
1/ Globally, statistics have to be revised. The authors have to include a "statistical analysis" section in the Material and Methods to explain how they proceeded and specify for each panel in the figure legend which tests they used according to the general rules of statistics.
2/ The setup of each experiment is confusing and needs to be detailed. Cell numbers are not coherent from one experiment to the other. As an example, there are discrepancies between Fig1 and Fig2. Based on the setup of the experiment in Fig.2 (Injection of B-ALL to mice followed by 2 injections of treatment every 5 days), mice have probably been sacrificed 12-14 days post leukemic cell injection. However, according to Fig.1, B cells and erythroid cells at this time point should be decreased >10 times while they are only decreased 2-4 times in Fig.2. This is also the case in Fig.4B-J or Fig.5D with even a lower decrease in B cells and erythroid cells despite a high number of leukemic cells. Please explain and give the end point for each experiment in each figure (main and supplemental).
3/ To formally prove that the observed effect is really due to LTab/LTbR signaling, the authors must perform further control experiments. LTbR signaling is better known for its positive role on lymphocyte migration. They cannot rule out by blocking LTbR signaling, that they inhibit homing of leukemic cells into the bone marrow through a systemic/peripheral effect, more than through an impaired crosstalk with BM LepR+ cells. They must confirm for inhibited/deficient LTbR signaling conditions, as compared to control, that similar B-ALL numbers home to the BM parenchyma at an early time point after injection. Furthermore, they cannot exclude that the effect on the expression of IL7 (and other genes), and consequently the effect on B cell numbers, is not simply due to the tumor burden. Indeed, B-ALL numbers/frequencies are different between control and inhibited/deficient signaling conditions at the time of analysis. The analyses should thus be performed at similar low and high tumor burden in the BM for both control and inhibited/deficient LTbR signaling conditions.
4/ LT/LTbR signaling is particularly known for its capacity to stimulate Cxcl12 expression. How do the authors explain that they see the opposite?
5/ The authors show that CXCL12 stimulates LTa expression in their cell line. They then propose that CXCR4 signaling in leukemic cells potentiates ALL lethality by showing that a CXCR4 antagonist reverses the decrease in IL7 and improves survival of the mice. This experiment is difficult to interpret. CXCL12 has been shown to be important for migration/retention of B-ALL in the BM and the decreased tumor burden is probably linked to a decreased migration more than an impaired crosstalk with LepR+ cells (see also point 3). If CXCL12 increases LTab expression, CXCR4 blockade should do the opposite. This result should be presented. The contradiction is that if B-ALLs induce a decrease in CXCL12 in the BM (in addition to IL7) and that CXCL12 regulates LTab levels, leukemic cells should be exhausted. Similarly, IL7 has been previously shown to stimulate LTab expression and B-ALL cells express the IL7R. Again, a decrease in IL7 should be unfavorable to B-ALL. How do they explain these discrepancies?
6/ In Supp 4A, RAG-/- mice are blocked at the pro-B cell stage and do not have pre-B cells. Please compare LTa and LTb expression by Artemis deficient pre-B cell to wt pre-B cells. In this experiment, the authors show that similarly to B-ALL artemis-/- pre-leukemic pre-B cells express high levels of LTab and induce IL7 downmodulation. Using mice deficient for LTbR in LepR+ cells, they show that IL7 expression is increased. However, in opposition to leukemic cells (see Figure 4F), pre-leukemic cells are increased in absence of LTab/LTbR signaling. Please explain this discrepancy. The authors use only one B-ALL model cell line for their demonstration (BCR-ABL expressing B-ALL). Another model should be used to confirm whether LTab/LTbR signaling does favor leukemic/pre-leukemic B cell growth.
7/ Pre-B cells are composed of large pre-B cells (pre-BCR+) and small pre-B cells (pre-BCR-). BCR-ABL B-ALL cells express the pre-BCR. What is the level of expression of LTa and LTb by each of these 2 subsets as compared to BCR-ABL B-ALL?
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