[Practical Case Sharing] Elucidation of the significance of lysophospholipid metabolism in cancer stem cells using genome-edited knockout mice (Dr. Naka, Hiroshima University)
Summary: Associate Professor Kazuhito Naka of Hiroshima University’s Research Institute for Radiation Biology and Medicine commissioned Setsuro Tech Inc. (Setsurotech) to produce Gdpd3 knockout mice in April 2018, and Setsuro Techdelivered three F0 mice in September of that year through an external breeder. Later, on September 17, 2020, the results of the analysis using Gdpd3 knockout mice were published in the British online scientific journal “Nature Communications”. We interviewed Assoc, Prof. Naka about the background of his research and its prospects.
Relapse of chronic myelogenous leukemia and cancer stem cells
Chronic myelogenous leukemia (CML) was once an incurable disease. However, the outcome of the treatment has dramatically improved after the approval of tyrosine kinase inhibitors (TKI: tyrosine kinase inhibitor) in 2001 in Japan, which targets the causative gene of CML, BCR-ABL1 .On the other hand, the prevention of relapse after TKI treatment is still a remaining issue. For example, suspension of the administration of imatinib, one of TKIs, in CML patients who are in remission showed only about half of the patients are reported to be cured and the rest to have relapsed.
In recent years, it has been known that CML stem cells are responsible for the recurrence of CML. CML stem cells can remain in a “dormant” state with low proliferative activity, thereby acquiring TKI resistance despite its production capability of a large number of CML cells. TKIs are considered less effective on CML stem cells due to their low proliferative activity, while TKIs are effective against CML cells with high proliferative activity. Therefore, it is believed that CML stem cells continue to survive, leading to the relapse of CML even after the TKI treatment. The team of Assoc. Prof. Naka has been researching a novel therapeutic strategy targeting CML cells in which the investigation of the mechanisms involved in the survival and dormancy of CML stem cells has been carried out.
Expression of lysophospholipid enzymes is upregulated in CML cancer stem cells
Assoc. Prof. Naka told us, “we have previously constructed a mouse model of CML stem cells . We isolated CML stem cells from mice with CML and conducted the gene expression profile analysis of genes whose expression was altered in CML stem cells by RNA-Seq. We found that Gdpd3, which is involved in lysophospholipid metabolism, is strongly expressed in CML stem cells. In general, phospholipids that make up cell membranes have two fatty acids, whereas lysophospholipids have only one fatty acid, which makes them more hydrophilic than phospholipids, resulting in biological activity. Gdpd3 is known as an enzyme that hydrolyzes such lysophospholipids to produce lysophosphatidic acid (LPA). However, no report described the cause of high expression of Gdpd3 in CML stem cells the role of lysophospholipids. Around that time, a sales representative of Setsuro Tech visited our laboratory and immediately decided to create Gdpd3 knockout mice. This serendipity initiated our new journey studying the function of Gdpd3 in CML stem cells.”
Cancer stem cells derived from Gdpd3 Knockout mice are not capable of remaining dormant
Assoc. Prof. Naka continued, “in the beginning, there were no noticeable abnormalities in the Gdpd3 knockout mice. However, further investigation was still essential in the study of very few stem cells. The more important molecules that carry out specific functions in a small number of stem cells, the more difficult it is to find their superficial characteristics.
It also implies that if we can find a hidden mechanism that is specific to cancer stem cells, we would reach CML treatment with fewer adverse effects. We immediately started to evaluate the function of CML stem cells by serial transplantation of CML stem cells. We took hematopoietic stem cells from the Gdpd3 knockout mice and wild-type mice as a comparison and introduced them with the BCR-ABL1 gene (primary transplantation). We expected that the CML stem cells from the Gdpd3 knockout mice were in a lack of function of Gdpde3 gene and would not develop CML. However, the results were just the opposite: CML stem cells from Gdpd3 knockout mice were more capable of developing leukemia than CML stem cells from wild-type mice, and all mice of Gdpd3 knockout mice had died. This result suggested that CML stem cells derived from Gdpd3 knockout mice were unable to remain dormant and had increased proliferative activity, ending up unable to maintain their stemness. We were shocked by this finding, and this experience was like being struck by lightning. It was a moment when we learned that nature was far greater than we thought. To demonstrate our hypothesis was true, we then purified the CML stem cells from the primary transplanted mice and transplanted them into other wild-type mice (a second transplant), finding, as we expected, the CML stem cells from the Gdpd3 knockout mice showed almost complete loss of the capability of developing CML. Indeed, our examination of the proliferative capacity of the CML stem cells showed a significant number of BrdU-positive cells in the S phase. These results demonstrate that the loss of the Gdpd3 gene increases the proliferative capability of CML stem cells and prevents them from remaining dormant.
In addition, we also found LPA production, activation of the AKT/mTORC1 pathway that promotes cell proliferation, and the decrease in Foxo3a activity involved in stem cell maintenance.
Finally, we assessed TKI resistance in the Gdpd3 -deficient CML stem cells, showing that dasatinib (TKI) administration significantly suppressed CML relapse in mice transplanted with CML stem cells derived from Gdpd3 knockout mice. These results indicated that Gdpd3-mediated lysophospholipid metabolism played an essential role in maintaining CML stem cells and that Gdpd3 deficiency caused the inability of CML stem cells to remain dormant, resulting in the reduced TKI resistance.
Assoc. Prof. Naka said, “the majority of CML cells are known to acquire proliferative capability through oncogene-dependent mechanisms such as BCR-ABL1. Anticancer drug therapy is effective in suppressing this proliferative activity. However, CML stem cells, the cause of relapse, can keep a dormant state through an oncogene-independent mechanism, making anticancer therapy difficult. In this study, we have elucidated a part of the dormancy mechanism of CML stem cells through the Gdpd3-mediated lysophospholipid metabolism pathway. We expect the development of molecularly targeted drugs that inhibit Gdpd3 and its administration with TKIs to improve the prognosis of CML patients.
Furthermore, we plan to carry out a functional analysis of Gdpd3-mediated lysophospholipid metabolism in cancer stem cells of other cancers. We hope that our research contributes to the development of cancer recurrence treatment with a new concept of targeting oncogene-independent lysophospholipid metabolism.”
Expectations to Setsurotech
Assoc. Prof. Naka told us, “we ordered the production of Gdpd3 knockout mice and Setsuro Tech delivered in a short delivery time, just four months, which enabled us to work on the analysis in a short period. This short delivery time should have realized with the high-quality work of people in Setsuro Tech and their sincere response to the order. Knockout mice are of irreplaceable value, and we will keep working on our research.
We also believe that the establishment of genetically modified mice is an area where genome editing technology shows the highest value. We hope that many scientists think about using genetically modified mice in their research and knockout mice provided by Setsuro tech get more widely used. We will ask Setsurotech to produce and deliver knockout mice for our next experiment as well.
Assoc. Prof. Naka talked about the advantage of using knockout mice in the functional analysis of a very small number of cancer stem cells. Although knockout mice are often used for phenotypic observation, his unique approach to isolating genetically deficient cancer stem cells to create a mouse disease model was quite impressive.
Naka K, Ochiai R, Matsubara E, et al. The lysophospholipase D enzyme Gdpd3 is required to maintain chronic myelogenous leukaemia stem cells. Nat Commun. 2020;11(1):4681.
Kantarjian H, O’Brien S, Jabbour E, et al. Improved survival in chronic myeloid leukemia since the introduction of imatinib therapy: a single-institution historical experience. Blood. 2012;119(9):1981-1987.
Mahon FX, Rea D, Guilhot J et al. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010; 11: 1029-1035.
Naka K, Hoshii T, Muraguchi T, et al. TGF-b-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia. Nature. 2010;463(7281):676-680.
Dr. Naka is an Associate Professor at the Hiroshima University Research Institute for Radiation Biology and Medicine, and received his PhD in Pathology from the Hiroshima University Graduate School of Medicine in 2000. He was a postdoctoral fellow at the National Institute for Longevity Sciences, an assistant professor at the Graduate School of Medical and Dental Sciences, Okayama University, an assistant professor at the Institute for Cancer Research, Kanazawa University, and an associate professor at the same institute before assuming his current position in 2015. His area of expertise is stem cell biology.