Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation
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Evaluation Summary:
FLT3 (Fms Related Receptor Tyrosine Kinase 3) activation occurs in a subset of acute myeloid leukemia (AML) cases and is associated with poor prognosis. This work is focused on the mechanisms of resistance to FLT3 inhibitors in AML. The authors show that the combination of the FLT3 inhibitor and an mTORC1 inhibitor reduces tumor burden and prevents relapse in FLT3 mutant AML. This paper is of interest in scientists and physicians investigating AML as well as scientists studying signaling pathways.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance toward improving therapeutic outcomes. In this study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn upregulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance and a promising strategy for improved treatment of FLT3 mutant AML patients.
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Author Response
Reviewer #1 (Public Review):
- Can the primary cells in Figure 2E and AML#1 and AML#2 be studied for mTORC1 activity by Western, as in 2D?
For reasons that we do not understand, we have been unable to effectively culture primary FLT3-ITD AMLs, despite being able to culture most other AMLs for weeks. This issue has prevented us from being able to perform biochemical analyses of FLT3-ITD AMLs in response to FLT3 inhibition.
- Additional genetic information should be provided if possible for the primary AML cells - what other mutations in addition to FLT3 were present? Were there any mTOR pathway alterations?
We provided the other mutations of AML#1 sample (NPM1 mutation) in the section METHODS-Therapeutic modeling in mice, as well as Figure legends 2E and 3D. There were no evident alterations in the mTOR pathway (beyond …
Author Response
Reviewer #1 (Public Review):
- Can the primary cells in Figure 2E and AML#1 and AML#2 be studied for mTORC1 activity by Western, as in 2D?
For reasons that we do not understand, we have been unable to effectively culture primary FLT3-ITD AMLs, despite being able to culture most other AMLs for weeks. This issue has prevented us from being able to perform biochemical analyses of FLT3-ITD AMLs in response to FLT3 inhibition.
- Additional genetic information should be provided if possible for the primary AML cells - what other mutations in addition to FLT3 were present? Were there any mTOR pathway alterations?
We provided the other mutations of AML#1 sample (NPM1 mutation) in the section METHODS-Therapeutic modeling in mice, as well as Figure legends 2E and 3D. There were no evident alterations in the mTOR pathway (beyond the FLT3-ITD mutation).
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Evaluation Summary:
FLT3 (Fms Related Receptor Tyrosine Kinase 3) activation occurs in a subset of acute myeloid leukemia (AML) cases and is associated with poor prognosis. This work is focused on the mechanisms of resistance to FLT3 inhibitors in AML. The authors show that the combination of the FLT3 inhibitor and an mTORC1 inhibitor reduces tumor burden and prevents relapse in FLT3 mutant AML. This paper is of interest in scientists and physicians investigating AML as well as scientists studying signaling pathways.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The proposed mechanism, involving a "novel pathway mediated by ATM and mTOR" appears to be preliminary, and not fully supported by the Western blots in Figure 2. The majority of the mechanistic experiments are done in a single cell line, further limiting the certainty of the mechanism.
Major comments:
- Figure 1A/B show two FLT3 mutant cell lines, but subsequent mechanistic experiments are primarily done in only the MOLM13 cells. IN Figure 1B, the effect of the CM on the MV411 cells is much less striking than on the MOLM13 cells, and the fact that these cells are omitted from the mechanistic experiments is a concern.
- The conclusion (and title of Figure 2) that the CM "reverses mTOR pathway suppression" is not fully supported by Figure 2D. The phosphorylation of mTOR at S2448 (which is a somewhat nonspecific …
Reviewer #1 (Public Review):
The proposed mechanism, involving a "novel pathway mediated by ATM and mTOR" appears to be preliminary, and not fully supported by the Western blots in Figure 2. The majority of the mechanistic experiments are done in a single cell line, further limiting the certainty of the mechanism.
Major comments:
- Figure 1A/B show two FLT3 mutant cell lines, but subsequent mechanistic experiments are primarily done in only the MOLM13 cells. IN Figure 1B, the effect of the CM on the MV411 cells is much less striking than on the MOLM13 cells, and the fact that these cells are omitted from the mechanistic experiments is a concern.
- The conclusion (and title of Figure 2) that the CM "reverses mTOR pathway suppression" is not fully supported by Figure 2D. The phosphorylation of mTOR at S2448 (which is a somewhat nonspecific indicator of actual mTOR activity) is higher at the start with the CM, which is not surprising given the many growth factors and nutrients in the CM and is suppressed by Quizartinib. Phospho-S6, which is a more specific indicator of mTORC1 activity is similar with and without CM. These experiments should be done in triplicate, with statistics, and other indicators of mTOR activity included (pS6K, p4EBP1) along with the total protein levels for all of the phospho-proteins including S6. These experiments should also be done in the MV411 cells.
- In Figure S3 (AML#2), everolimus alone nearly completely suppressed the leukemic cells in the blood, consistent with the possibility that it has actions that are unrelated to Quizartinib.
- In Figure 3G, (AML#1) why is there no vehicle data for the 21 Day treatment time point? What was the leukemia burden in the vehicle? Without this, the data are hard to interpret. It appears that the combination is not different from Quiz alone. IN Figure 3E, at the earlier time point, it appears that everolimus inhibits the leukemia burden although this did not reach significance, and it also appears that the combination is not different from Quizartinib alone.
- Similarly, the title of Figure 4 is "Restoration of mTOR signaling by hBMSC-CM ..." but the authors have not convincingly demonstrated that mTOR signaling is being "restored" - instead this could be a combined effect of mTORC1 inhibition and Quizartinib that is not mechanistically directly linked.
- Figure 4 again relies on a single cell line MOLM13.
- Can the primary cells in Figure 2E and AML#1 and AML#2 be studied for mTORC1 activity by Western, as in 2D?
- Additional genetic information should be provided if possible for the primary AML cells - what other mutations in addition to FLT3 were present? Were there any mTOR pathway alterations?
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Reviewer #2 (Public Review):
The manuscript by Hae J. Park et al reports up-regulation of oxidative phosphorylation due to the activation of the mTOR signaling pathway is of critical importance to the therapeutic resistance of FLT3 mutant AML cells in a bone marrow environment. A combination of treatments with mTOR inhibitor and FLT3 inhibitor drastically eliminates all the marrow resident AML cells. Although the basic findings have been reported by other groups, they have potential clinical relevance that might benefit AML patients with FLT3-ITD mutations. Along these lines the manuscript is of interest, however important critiques need to be addressed with additional experimental data/revision.
1. Most of the mechanism implies mTOR-dependent translation control, similar to Sonenberg's findings as cited in the manuscript. However, data …
Reviewer #2 (Public Review):
The manuscript by Hae J. Park et al reports up-regulation of oxidative phosphorylation due to the activation of the mTOR signaling pathway is of critical importance to the therapeutic resistance of FLT3 mutant AML cells in a bone marrow environment. A combination of treatments with mTOR inhibitor and FLT3 inhibitor drastically eliminates all the marrow resident AML cells. Although the basic findings have been reported by other groups, they have potential clinical relevance that might benefit AML patients with FLT3-ITD mutations. Along these lines the manuscript is of interest, however important critiques need to be addressed with additional experimental data/revision.
1. Most of the mechanism implies mTOR-dependent translation control, similar to Sonenberg's findings as cited in the manuscript. However, data presented that the OXPHOS gene expression also changes in some experiments. Is this consistent among the different experimental settings? If this is the case, are these changes in gene expression consistent with PGC1s or ERRs OXPHOS target genes that are controlled via mTOR, see Cunningham et al. Nature 2007 Nov 29;450(7170):736-40 or Dufour et al. Cell Rep. 2022 Mar 22;38(12):110534-
2. The authors used hBMSC conditioned medium to mimic the bone marrow environment and used normal RPMI media as a control, to investigate the effects of bone marrow-released stromal factors on the TLT3 inhibitor resistance of AML. However, in addition to the "stromal factors", conditioned medium has different pH, metabolites, glucose levels, and more compared to RPMI medium, making the system complicated. Can the authors use the conditioned medium from HEK-293 or a spleen cell line as the control?
3. The treatment times of drugs changed from experiment to experiment. Is there any reason for this?
4. The quality of western blots looks over-adjusted. This is a very poor imaging quality with all blots being very pixel with non-quantitative adjustment. Please see other journals for quality blots on mTOR signaling. Antibodies for this pathway are of very good quality, these adjustment of images is not rigorous.
5. There are some problems with ATM, mTOR, translation axis, and connection. In Fig 7C, knockdown of ATM completely eliminates mTOR but the translation indicators, p-4EBP1, and p-S6 are kept unchanged, indicating inhibition of mTOR in these cells has no effect on protein translation in general, which is totally against the data shown previously in this manuscript.
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Reviewer #3 (Public Review):
In patients with AML and FLT2 ITD mutations, inhibiting FLT3 frequently reduces the number of leukemic cells in the peripheral blood, while the number of leukemic blasts in the marrow remains unchanged. The mechanisms behind this differential response remain uncertain.
In this paper, Park et al., show that the mTOR pathway is enriched in AML cells exposed to stroma compared to cells not exposed to the stroma. Combining mTOR and FLT3 inhibitors kills AML cells in the presence of stroma. As such, this paper offers potential mechanisms to explain the clinical problem of why FLT3 inhibitors frequently fail to eradicate marrow blasts while eliminating blasts in the peripheral blood. In addition, this paper provides the rationale for a future clinical trial combining FLT3 and mTOR inhibitors in patients with FLT3 …
Reviewer #3 (Public Review):
In patients with AML and FLT2 ITD mutations, inhibiting FLT3 frequently reduces the number of leukemic cells in the peripheral blood, while the number of leukemic blasts in the marrow remains unchanged. The mechanisms behind this differential response remain uncertain.
In this paper, Park et al., show that the mTOR pathway is enriched in AML cells exposed to stroma compared to cells not exposed to the stroma. Combining mTOR and FLT3 inhibitors kills AML cells in the presence of stroma. As such, this paper offers potential mechanisms to explain the clinical problem of why FLT3 inhibitors frequently fail to eradicate marrow blasts while eliminating blasts in the peripheral blood. In addition, this paper provides the rationale for a future clinical trial combining FLT3 and mTOR inhibitors in patients with FLT3 mutated AML.
Strengths of the paper include the use of primary AML samples and extension into mouse models. Chemical and genetic approaches are used to validate some key findings.
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