L1 drives HSC aging and affects prognosis of chronic myelomonocytic leukemia

Ju, Z. et al. Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment. Nat. Med. 13, 742–747 (2007). CAS Article Google Scholar De Cecco, M. et al. Genomes of replicatively senescent cells undergo global epigenetic changes leading to gene silencing and activation of transposable elements. Aging Cell. 12, 247–256 (2013). Article Google Scholar Murnane, J. P. & Sabatier, L. Chromosome rearrangements resulting from telomere dysfunction and their role in cancer. Bioessays 26, 1164–1174 (2004). CAS Article Google Scholar Niyongere, S. et al. Heterogeneous expression of cytokines accounts for clinical diversity and refines prognostication in CMML. Leukemia 33, 205–216 (2019). CAS Article Google Scholar Download references We thank the Dr. Jian Mao (School of Medicine, Hangzhou Normal University) for technical assistance. This work was supported by Grants 2016YFA0100602, 2017YFA0103302, 2018YFA0109300 from the National Key Research and Development Program of China; Grants 81525010, 91749203, 81871116, 81501214, 91749117, 81770155, and 81771502 from the National Natural Science Foundation of China; Grants LQ14C070002 from the Natural Science Foundation of Zhejiang Province of China; Grant 2018GZR110103002 from Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory and Grant 2017ZT07S347 from the Program for Guangdong Introducing Innovative and Enterpreneurial Teams. This work was supported by the Science Foundation for Distinguished Young Scholars of Guangdong Province (2019B151502008) to Hu Wang. These authors contributed equally: Ying Wang, Jin-ping Zheng Key Laboratory of Aging and Cancer Biology of Zhejiang Province, Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, 311121, China Ying Wang, Junyi Wang, Lingjie Xu, Hu Wang & Zhenyu Ju Department of Public Health and Preventive Medicine, Changzhi Medical College, Changzhi, Shanxi, 046000, P. R. China Jin-ping Zheng Key Laboratory of Regenerative Medicine of Ministry of Education, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, 510632, China Ying Luo, Hu Wang & Zhenyu Ju CAS Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China Jinyong Wang Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, 02903, USA John M. Sedivy Department of Colorectal Surgery, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, China Zhangfa Song You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar Y.W., JP.Z., H.W., Y.L., JY.W., and LJ.X. carried out the experiments. Y.W., JP.Z., H.W., JinY W., J.M.S., ZF.S., and ZY.J. analyzed the data. Y.W., JP.Z., H.W., J.M.S., and ZY.J. wrote the paper. Correspondence to Ying Wang or Zhangfa Song or Hu Wang or Zhenyu Ju. The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and Permissions Wang, Y., Zheng, J., Luo, Y. et al. L1 drives HSC aging and affects prognosis of chronic myelomonocytic leukemia. Sig Transduct Target Ther 5, 205 (2020). https://doi.org/10.1038/s41392-020-00279-4 Download citation Received: 26 March 2020 Revised: 27 July 2020 Accepted: 30 July 2020 Published: 19 September 2020 DOI: https://doi.org/10.1038/s41392-020-00279-4

To further illustrate the critical function of L1 in regulating cGAS signaling and HSC function, we administered the L1 reverse transcription inhibitor 3TC to G3Terc −/− mice ( Supplementary Fig.  S3a). We found that the cytosolic accumulation of L1 cDNA (Fig. 1f, Supplementary Fig. S3b), the expression of 2'3'-cGAMP (Fig. 1g), and the phosphorylation levels of TBK1, IRF3, and NF-κB p65 were significantly decreased in G3Terc −/− mice treated with 3TC ( Supplementary Fig. S3c), but the cytosolic accumulation of L1 cDNA and the cGAS signaling was not affected in WT mice treated with 3TC ( Fig. 1f-g, Supplementary Fig. S3b, d). Consequently, the cytokines production were dramatically reduced in BM cells and plasma of G3Terc −/− mice treated with 3TC, but not in that of WT mice treated with 3TC (Fig. 1h, Supplementary Fig. S3e, f). Furthermore, Depletion of L1 significantly decreased cytokines expression in BM cells of G3Terc −/− mice (Fig. 1i). To verify whether reduced inflammation by suppression of L1 could rescue the impaired HSC maintenance and function in telomere dysfunctional mice as we previous reported, 1 we performed flow cytometry analysis and the results showed that 3TC treatment could recover the frequency of long-term HSCs (LTs) and multipotential progenitors (MPPs) in telomere dysfunctional mice, and 3TC treatment had no effect on the proportion of HSCs in WT mice (Fig. 1j, Supplementary Fig. S4a). Notably, competitive transplantation experiment revealed a significant improvement in the repopulating capacity of HSCs isolated from G3Terc −/− mice treated with 3TC, but without affected the lineage distribution at steady state ( Fig. 1k-l, Supplementary Fig. S4b). Together, these results demonstrate that suppression of L1 alleviates inflammation, thereby improves the HSC maintenance and function.
In human, chronic myelomonocytic leukemia (CMML) is a chronic myeloid neoplasm of the elderly with a poor prognosis. Mice harboring an oncogenic G12D mutation in Nras locus (Nras G12D mice) can develop a CMML-like disease. Recent study reported that inflammatory cytokines are elevated in CMML patients. 4 To determine whether the telomere dysfunction induced inflammatory environment affects the prognosis of leukemia, we established a mouse BM transplantation model by using the BM from Nras G12D mice as donor, and half lethal dose irradiated G3Terc −/− or WT mice as recipients ( Supplementary Fig.  S5a). We found that both G3Terc −/− and WT mice transplanted with Nras G12D BM (G3Terc −/− recipient mice and WT recipient mice) could develop a CMML-like disease, and observed that diseased mice showed enhanced hepatosplenomegaly compared to control mice ( Supplementary Fig. S5b, c), and gradually increased frequency of myeloid cells in peripheral blood (PB) from both WT recipient mice and G3Terc −/− recipient mice ( Supplementary Fig. S5d). Notably, the G3Terc −/− recipient mice showed significantly reduced survival compared with WT recipient mice (Fig. 1m). The white blood cells count and the frequency of neutrophils were significantly increased, whereas the red blood cells count, platelets count, and frequency of lymphocytes were decreased in G3Terc −/− recipient mice ( Supplementary Fig. S5e). The BM analysis showed that absolute number of T and B lymphocytes were significantly decreased in G3Terc −/− recipient mice (Fig. 1n). The absolute number and frequency of recipientderived LTs, megakaryocytic/ erythroid progenitors (MEPs) and common lymphoid progenitors (CLPs) were decreased in G3Terc −/− recipient mice, and the absolute number and frequency of donor-derived common myeloid progenitors (CMPs), MEPs and CLPs were decreased in G3Terc −/− recipient mice as well (Fig. 1o, p, Supplementary Fig. S5f-h). In addition, we found that the cytokines production were dramatically upregulated in plasma of G3Terc −/− recipient mice, although IL-6 was also increased in donor mice ( Supplementary Fig. S5i). Together, these results indicate that the repression of the normal hematopoiesis and increased inflammation contributes to the decreased survival of G3Terc −/− recipient mice.
To determine whether improvement of HSC by L1 inhibition is beneficial in prognosis of CMML, we administered 3TC to G3Terc −/− recipient mice and WT recipient mice ( Supplementary  Fig. S6a), and found that the 3TC treatment extended the survival of G3Terc −/− recipient mice, but not WT recipient mice (Fig. 1q).
The expression of cytokines in plasma of G3Terc −/− recipient mice treated with 3TC were significantly decreased (Supplementary Fig.  S6b). Interestingly, we found that 3TC could not relieve the hepatosplenomegaly, high proportion of myeloid cells in PB and high absolute number of myeloid cells in BM from G3Terc −/− Letter recipient mice (Fig. 1r, Supplementary Fig. S6c-e), and the absolute number and frequency of donor-derived hematopoietic stem/ progenitor cells (HSPCs) were comparable between 3TC treated and untreated G3Terc −/− recipient mice (Fig. 1s, Supplementary  Fig. S6f). Notably, the absolute number and frequency of recipientderived LTs, MEPs and megakaryocyte progenitors (MKPs) were dramatically increased, and the PLTs count was rescued to normal level in G3Terc −/− recipient mice with 3TC treatment (Fig. 1t,  Supplementary Fig. S6g, h). To explore whether 3TC treatment also contributes to the repression of donor cell (Nras G12D ), we checked the mRNA expression and CpG methylation level of L1 in diseased mice. The results showed decreased CpG methylation at L1 promoter region in endogenous G3Terc −/− BM cells, but not in endogenous WT BM cells, the donor-derived (Nras G12D ) BM cells in WT and G3Terc −/− recipient mice (Supplementary Fig. S7a). The expression level of L1 in different groups were consistent with the CpG methylation levels ( Supplementary Fig. S7b). Altogether, these results indicate that the suppression of L1 by 3TC treatment in CMML mice reduces telomere dysfunction induced inflammation and attenuates the impaired hematopoiesis, thereby extended survival of the diseased mice, probably due to the improvement of HSPCs maintenance.
In summary, we found that L1 activation is responsible for the cGAS signaling induced inflammatory responses in G3Terc −/− mice. 3TC treatment attenuated the aging-associated decline of HSC maintenance and function, thereby extended the survival of G3Terc −/− recipient mice transplanted with oncogenic Nras G12D BM. Our findings suggest that reverse transcriptase inhibition may serve as a new therapeutic strategy for patients suffering from age-related disorders.