The lysophospholipase D enzyme Gdpd3 is required to maintain chronic myelogenous leukaemia stem cells

Although advanced lipidomics technology facilitates quantitation of intracellular lipid components, little is known about the regulation of lipid metabolism in cancer cells. Here, we show that disruption of the Gdpd3 gene encoding a lysophospholipase D enzyme significantly decreased self-renewal capacity in murine chronic myelogenous leukaemia (CML) stem cells in vivo. Sophisticated lipidomics analyses revealed that Gdpd3 deficiency reduced levels of certain lysophosphatidic acids (LPAs) and lipid mediators in CML cells. Loss of Gdpd3 also activated AKT/mTORC1 signalling and cell cycle progression while suppressing Foxo3a/β-catenin interaction within CML stem cell nuclei. Strikingly, CML stem cells carrying a hypomorphic mutation of Lgr4/Gpr48, which encodes a leucine-rich repeat (LRR)-containing G-protein coupled receptor (GPCR) acting downstream of Gdpd3, displayed inadequate disease-initiating capacity in vivo. Our data showing that lysophospholipid metabolism is required for CML stem cell maintenance in vivo establish a new, biologically significant mechanism of cancer recurrence that is independent of oncogene addiction.

For Lipidomics data in Fig. 3a,b and Fig. 4a-c, the original data are available from Source Data file, and Supplementary Method1, 2, 3. For RNA sequencing data in Fig. 7a, and Supplementary Figure 1, 11a-c, our data are available from a public database gene expression omnibus (GEO, ID: GSE70031 and GSE149442, NCBI, NIH, USA) (https://www.ncbi.nlm.nih.gov/gds/) and Source Data file. Gene expression levels were measured with the bioconductor package DESeq2 v.1.20.0 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html) using the Ensembl database (https://ensembl.org/index.html). MA-plots were created using the Bokeh library (ver. 0.13.0) (https://docs.bokeh.org/en/0.13.0/). GO enrichment analyses were performed using the DAVID Bioinformatics Resource 6.8. (http://david.abcc.ncifcrf.gov). Gene expression data for Fig. 6d was downloaded from the public microarray dataset in the GEO database under accession code GSE12211 (https:// www.ncbi.nlm.nih.gov/gds/). The source data for 3a,b,Supplementary Figs. 1,3b,c,5a,b,7,13a,b,and Supplementary  We did not calculate sample size of mice before transplantation of CML stem cells into recipient mice in our experiments. For first-round transplantation in Fig.2a, Fig. 6a, Fig.7e, normal haematopoietic LSK cells isolated from wild-type and Gdpd3 KO, or wild-type and Lgr4 Gt/Gt mice were transduced with BCR-ABL1 oncogene, and approximately same numbers of BCR-ABL1-transduced LSK cells were transplanted into 5 to 8 recipient mice to compare the survival rate of CML-affected mice on the transplantation. The number of recipient mice transplanted with the LSK cells were different depend on the LSK cell number obtained from each mouse. Thus, different number of recipient mice transplanted with CML LSK cells were compared depend on the normal LSK cell number obtained. To reduce the mouse number, we examined minimal number of recipient mice on each experiment, and repeated transplantation experiments to examine whether or not we could establish statistical significance.
For second-round serial transplantation in Fig.2b, we isolated CML LSK cells from retro-CML mouse model. There was individual difference on the cell number of CML stem cells in each CML-affected mouse. (The maximum numbers of CML LSK cells were sorted from both wild-type and Gdpd3 KO retro-CML mice, and same number of these CML LSK cells were transplanted into recipient mice.) Thus, different number of recipient mice transplanted with CML LSK cells were compared depend on the CML stem cell number obtained. We repeated minimal number of transplantation experiments to examine whether or not we could establish statistical significance.
For the survival experiment of tet-CML mouse model in Fig.1g, the minimal number of mice was compared to examine whether or not we could establish statistical significance from wild type and Gdpd3-deficient tet-CML cohorts that were born.
If any recipient mouse that were transplanted with CML stem cells passed away within 10 days after irradiation due to acute radiation effects or technical failure of transplantation, we exclude the mouse from the cohort.
We repeated twice or three times experiments to obtain enough sample size and to confirm the results.
For the first-and second-round transplantations in Fig.2a and Supplementary Figs.5a,b,7,9,13b, recipient C57BL/6 and C57BL/6-CD45.1 mice were randomly allocated after one week of purchase, and were transplanted with normal and CML LSK cells. The normal and CML LSK cells were sorted from the age and sex-matched donor mice (i.e., wild-type, Gdpd3 KO, or Lgr4 Gt/Gt mice). For the experiments of normal and tet-CML mouse model in Fig.1b, d-g, Fig.3a, b,Fig.7a,c,d,Fig.8a,b,Supplementary Figs. 2a,b,3b,c,13a,14,15, cells were isolated from the age and sex-matched mice (i.e., wild-type, Gdpd3 KO, or Lgr4 Gt/Gt Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. We complied previous reports as followed ;Naka et al., Nature 463, 676-680, 2010., Reynaud et al., Cancer Cell, 20, 661-673, 2011, and Naka et al., Nature Communications, 6, 8039, 2015 nature research | reporting summary Note that full information on the approval of the study protocol must also be provided in the manuscript.

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OP-9 murine stromal cells and K562 human CML cells were authenticated.
Cell lines were not tested for mycoplasma contamination.
This study does not contain samples collected from fields.
We did not use filed-collected samples in this study.
All animal care and experimentation were carried out in accordance with the guidelines for animal and recombinant DNA experiments of Hiroshima University (Authorized Protocol Numbers A18-36, A18-37 and 30-257) and Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology Japan. The latest inspection by the Japanese Association for Laboratory Animal Sciences was at December 5th, 2017.
The viable BMMNCs were collected from a primary CML patient for diagnosis. The patient was confirmed diagnosis of chronic phase CML. The BMMNCs were isolated from a surplus of bone marrow fluid without any additional bone marrow aspiration to the patient, and the anonymized BMMNCs were stored in freezing condition.
The BMMNCs from a primary CML patient for diagnosis at the University Hospital, Dokkyo Medical University (Tochigi, Japan) were collected between October 2014 and March 2019.
Institutional Review Board (IRB) of the University Hospital, Dokkyo Medical University (Tochigi, Japan) (IRB approval number: 26058). All procedures involving human participants were performed in compliance with the relevant ethical standards.