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Letters to the Editor
leukin-1b, interleukin-6, tumor necrosis factor-α and CCL3 mRNA transcripts compared to controls (Figure 2I), suggesting that some of the effects of exosomes on adipocyte differentiation might be attributed indirectly to these pro-inflammatory cytokines (Online Supplementary Figure S3A-C) and perhaps may also be responsible for the differentiation block. This is the first study establish- ing a role for leukemic exosomes in the induction of adipocyte atrophy by upregulation of ATGL/HSL lipolytic enzyme activity, directly or through inflammatory cytokines.
As a proof of principle, we first cultured the adipocytes with cytokines, AML exosomes alone or in the presence of atglistatin (an ATGL inhibitor) in nutrient-deprived conditions for 48 h. AML cells cultured in these exo- some/cytokine conditioned media (CM) had lower apop- tosis, possibly by providing lipid substrates from adipocytes to drive mitochondrial b-oxidation (an alter- nate energy pathway) in leukemic cells during nutrient deficiency. However, atglistatin treatment increased apoptosis by blocking ATGL-mediated free-fatty acid (FFA) release (Figure 2J and K). The inhibition of FFA resulted in reduced leukemic fatty acid oxidation, evi- denced by reduced mitochondrial Cpt1a gene expression (Figure 3A; Online Supplementary Figure S3D).
Atglistatin or HSL inhibitor (CAY10499) treatment also reversed the leukemia CM-induced reductions in adipocyte size (Figure 3B), possibly by inhibiting exo- some-induced triglyceride breakdown. Since leukemia- induced adipocyte breakdown releases FFA into the sur- rounding tumor environment to drive the leukemic cells’ energy processes, we hypothesized that atglistatin or CAY10499 could inhibit FFA release and hence lower the uptake by leukemia cells. To test this hypothesis, we used adipocytes with fluorescently labeled FFA analogs and observed that atglistatin or CAY10499 treatment could significantly reduce BODIPY transfer in leukemic cells (Figure 3C).
Next, we evaluated the benefit of ATGL inhibition in the context of the resistance to chemo/radiotherapy induced by adipocytes and leukemic exosomes. MV4-11 cells were cultured in control or AML adipocyte CM together with or without atglistatin and 5 nM quizartinib (a tyrosine kinase inhibitor) in nutrient-deprived condi- tions. Compared to treatment with quizartinib alone, the atglistatin and quizartinib combination significantly reduced the survival advantage mediated by leukemia exosome-induced lipolysis, suggesting that atglistatin could be used as combination therapy with quizartinib to enhance its efficacy on AML cells by limiting nutrients released from adipocytes (Figure 3D).
Similarly, atglistatin also increased radiation-induced leukemia killing to overcome the adipocyte-induced resistance of leukemia to radiotherapy (Figure 3E). We further proved that leukemia-specific ATGL is essential for leukemia cell proliferation and showed that the pro- liferation could be inhibited pharmacologically by atglis- tatin in a dose-dependent manner (P=0.001 and P<0.0001 at day 4 at a doses of 50 mM and 100 mM atglis- tatin, respectively) (Figure 3F) or by shRNA-mediated genetic knockdown of the ATGL gene (P<0.0001 at day 4) (Figure 3G, Online Supplementary Figure S3E).
Since losses of osteoblasts and adipocytes are key fea- tures during leukemia progression, we hypothesized that increasing osteoblast or adipocyte content pharmacolog- ically with either zoledronic acid (osteoblasts) or GW1929 (adipocytes) might delay disease development. The treated mice stroma exhibited increased levels of Ocn and Pparg genes in BM stroma providing a functional val-
idation of the increases in osteoblast and adipocyte num- bers (Online Supplementary Figure S4A and B). Furthermore, zoledronic acid or GW1929 treatment in- vivo prolonged the survival of AML and ALL mice (Online Supplementary Figure S4C-E), confirming the earlier belief of a role of osteoblasts and adipocytes in assisting normal hematopoiesis to delay leukemia progression.
In conclusion, we discovered that leukemia induces defective preadipocyte expansion and describe a novel mechanism of leukemic exosome-induced adipocyte loss through the activation of lipolytic genes. Finally, leukemia- or exosome-induced niche reprogramming could be rescued by GW1929 and zoledronic acid to limit the progression of leukemia. This study further strength- ens our earlier observations of leukemia exosome- induced BM microenvironmental dysregulation to favor leukemia propagation. Taken together, these findings provide a strong rationale for targeting the defective niche with leukemia exosome inhibition and normalizing BM niche components with pharmacological agents in conjunction with conventional therapies.
Bijender Kumar,1,2* Marvin Orellana,1° Jamison Brooks,1 Srideshikan Sargur Madabushi,1 Paresh Vishwasrao,1 Liliana E. Parra,1 James Sanchez,3 Amandeep Salhotra,2,3 Anthony Stein,2,3 Ching-Cheng Chen,2 Guido Marcucci2,3 and Susanta Kumar Hui1
1Department of Radiation Oncology, Beckman Research Institute,City of Hope National Medical Center, 2HematologicalMalignancies, Stem Cell Transplantation Institute, City of Hope National Medical Center and 3Department of Hematology and HCT, City of Hope National Medical Center, Duarte, CA, USA
°Current address: Department of Stem Cell Transplantation and Cellular Therapy, MD Anderson Cancer Center, Houston, TX, USA
Correspondence:
BIJENDER KUMAR - bkumar1@mdanderson.org SUSANTA KUMAR HUI - shui@coh.org doi:10.3324/haematol.2019.246058
Received: January 9, 2020.
Accepted: September 4, 2020.
Pre-published: September 10, 2020
Disclosures: AS provides consulting for Amgen and Stemline and is on the speakers’ bureau for the former company. SKH receives hono- raria from and consults for Janssen Research & Development, LLC. All other authors declare that they have no competing financial interests.
Contributions: BK, MO, JB, SSM, PV and LEP performed experi- ment; BK and SKH conceived the idea; BK designed experiments, interpreted data and wrote the manuscript; AS, AS, GM and SKH provided the leukemia patients’ samples; SSM, JS, GM and SKH edited the manuscript. All authors read and approved the paper.
Acknowledgments: we would like to thank Dr Michael Kahn, Molecular Medicine, COH, for carefully reviewing the manuscript.
We also thank Donna Isbell of the Animal Resource Center for animal care, Analytical Cytometry Core for flow cytometry support and
Dr. Xiwei Wu from the Integrative Genomics Core laboratory for help with the single cell sequencing. Research reported in this publication also included work performed in the microscopy and pathology cores at City of Hope.
Funding: this study was supported by grants to SKH (1R01CA154491, 2R01CA154491), City of Hope Cancer
Center (P30CA033572) and American Cancer Society Grant 128766-RSG-15-162(CCC). The content is solely the responsibility of the authors and does not necessarily represent the official views
of the National Institutes of Health or other funding agencies.
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