Lnk deficiency enhances translesion synthesis to alleviate replication stress and promote hematopoietic stem cell fitness
Brijendra Singh, Md Akram Hossain, Xiao Hua Liang, Jeremie Fages, Carlo Salas Salinas, Roger A. Greenberg, Wei Tong
Brijendra Singh, Md Akram Hossain, Xiao Hua Liang, Jeremie Fages, Carlo Salas Salinas, Roger A. Greenberg, Wei Tong
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Research Article Development Hematology

Lnk deficiency enhances translesion synthesis to alleviate replication stress and promote hematopoietic stem cell fitness

Abstract

The adaptor protein LNK/SH2B3 negatively regulates hematopoietic stem cell (HSC) homeostasis. Lnk-deficient mice show marked expansion of HSCs without premature exhaustion. Lnk deficiency largely restores HSC function in Fanconi anemia (FA) mouse models and primary FA patient cells, albeit protective mechanisms remain enigmatic. Here, we uncover a role for LNK in regulating translesion synthesis (TLS) during HSC replication. Lnk deficiency reduced replication stress–associated DNA damage, particularly in the FA background. Lnk deficiency suppressed single-strand DNA breaks, while enhancing replication fork restart in FA-deficient HSCs. Diminished replication-associated damage in Lnk-deficient HSCs occurred commensurate with reduced ATR/p53 checkpoint activation that is linked to HSC attrition. Notably, Lnk deficiency ameliorated HSC attrition in FA mice without exacerbating carcinogenesis during aging. Moreover, we demonstrated that enhanced HSC fitness from Lnk deficiency was associated with increased TLS activity via REV1 and, to a lesser extent, TLS polymerase eta (η). TLS polymerases are specialized to execute DNA replication in the presence of lesions or natural replication fork barriers that stall replicative polymerases. Our findings implicate elevated use of these specialized DNA polymerases as critical to the enhanced HSC function imparted by Lnk deficiency, which has important ramifications for stem cell therapy and regenerative medicine in general.

Authors

Brijendra Singh, Md Akram Hossain, Xiao Hua Liang, Jeremie Fages, Carlo Salas Salinas, Roger A. Greenberg, Wei Tong

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Figure 7

Lnk-deficient HSCs are sensitive to REV1 inhibition, and their superior reconstituting activity depends on REV1-mediated TLS.

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Lnk-deficient HSCs are sensitive to REV1 inhibition, and their superior...
(A) BM cells from WT and Lnk–/– mice were plated in semi-solid methylcellulose culture containing a graded dose of REV1i in triplicate, at 30,000 and 15,000 cells per plate, respectively. Total (left) and relative (right) colony-forming capacities are shown. Mean values and SD are shown. P values were calculated using 2-way ANOVA; ****P < 0.0001. (B) Schematic illustration of the lentiviral transduction/BMT experimental scheme. (C) SLAM LSK HSCs from WT and Lnk–/– mice were sorted into 96-well plates. Cells were cultured in DMSO or 5 μM REV1i for 3 days; then cells from each well were transplanted into each lethally irradiated recipient mouse. Donor percentages from each group in the peripheral blood are shown. Each symbol represents an individual mouse. Horizontal lines indicate mean frequencies, and error bars indicate SD. P values were calculated using 1-way ANOVA; **P < 0.01, ***P < 0.001, ****P < 0.0001.