Schlafen 11 (SLFN11), a restriction factor for replicative stress induced by DNA-targeting anti-cancer therapies

Abstract

Schlafen 11 (SLFN11) sensitizes cells to a broad range of anti-cancer drugs including platinum derivatives (cisplatin and carboplatin), inhibitors of topoisomerases (irinotecan, topotecan, doxorubicin, daunorubicin, mitoxantrone and etoposide), DNA synthesis inhibitors (gemcitabine, cytarabine, hydroxyurea and nucleoside analogues), and poly(ADPribose) polymerase (PARP) inhibitors (olaparib, rucaparib, niraparib and talazoparib). In spite of their different primary mechanisms of action, all these drugs damage DNA during S-phase, activate the intra-S-phase checkpoint and induce replication fork slowing and stalling with single-stranded DNA segments coated with replication protein A. Such situation with abnormal replication forks is known as replication stress. SLFN11 irreversibly blocks replication in cells under replication stress, explaining why SLFN11-positive cells are markedly more efficiently killed by DNA-targeting drugs than SLFN11-negative cells. SLFN11 is inactivated in ~50% of cancer cell lines and in a large fraction of tumors, and is linked with the native immune, interferon and T-cells responses, implying the translational relevance of measuring SLFN11 expression as a predictive biomarker of response and resistance in patients. SLFN11 is also a plausible epigenetic target for reactivation by inhibitors of histone deacetylases (HDAC), DNA methyltransferases (DNMT) and EZH2 histone methyltransferase and for combination of these epigenetic inhibitors with DNA-targeting drugs in cells lacking SLFN11 expression. In addition, resistance due to lack of SLFN11 expression in tumors is a potential indication for cell-cycle checkpoint inhibitors in combination with DNA-targeting therapies.

Keywords: ATR; Cell cycle checkpoint; DNA damage response; DNA-targeting agent; Drug resistance; PARP inhibitors; Replication stress; SLFN11; Schlafen 11; Topoisomerases.

Figure 1.

Scheme of the direct sites of drug action and pharmacological targets: deoxynucleotide triphosphates (dNTPs), chain elongation, DNA polymerases (POL), DNA template alterations, cyclin-dependent kinases (CDK1/2) and cell cycle checkpoints (Chk1 and Chk2). Representative drugs used in the clinic or as research tools are listed for each target. Ori: replication origin, 6-MercaptoP: 6-mercaptopurine, and UCN-01: 7-hydroxystaurosporine.

Figure 2.

Mechanisms of action of DNA-targeting agents that induce replication stress. Representative drugs are listed in each panel. A-D: Drugs targeting the DNA template. A: Platinums generate DNA interstrand-crosslink (red-cross). B and C: Topoisomerase I and II inhibitors (TOP1i and TOP2i, gray boxes) bind at the enzyme-DNA interface in the break sites and block the re-ligation of the TOP-DNA cleavage complexes (TOPcc). D: PARP inhibitors (PARPi) bind the catalytic pocket of PARP1 and PARP2 and trap PARP1 and PARP2 on the DNA, generating toxic PARP-DNA complexes. E: DNA elongation is blocked by reducing dNTP pools or inhibition of DNA polymerases. F: Under replication stress by various types of drugs, replication protein A (RPA) binds single-strand DNA and forms polymer where ATR is recruited and activated. RPA filaments also recruit SLFN11. G: The 5’-ends of DSB generated by fork collision are resected by the MRN (MRE11, RAD50, NBS1) complex and CtIP (RBBP8), and by DNase2 and EXO1. The 3’-single-stranded DNA tails are coated by RPAs, where ATR is recruited and activated.

Figure 3.

Scheme of the domain structure of SLFN11 (901 amino acids). SLFN11 has two main domains: 1/ the N-terminus nuclease domain is highly conserved with SLFN13, which acts as ribonuclease for t-RNA (Yang, et al., 2018); 2/ the C-terminus helicase domain contains Walker A and Walker B motifs and the nuclear localization signal (NLS). The point mutation at E669 into Q669 disrupts the function of SLFN11 for chromatin opening and drug sensitization, yet SLFN11-E669Q retains chromatin binding ability (J. Murai, et al., 2018). The RPA binding domain is essential for SLFN11 recruitment to chromatin in response to DNA damage (Mu, et al., 2016). The SLFN box and SWADL domains are conserved across all human SLFNs (5, 11, 12, 13 and 14), although their functions are not characterized.

Figure 4.

Molecular model of SLFN11-induced replication fork block in response to replication stress at early time points (A, B) and later time points (C, D). A: In SLFN11-expressing cells, both ATR and SLFN11 are recruited to stressed replication forks by replication protein A (RPA)-coated single-stranded DNA. SLFN11 also binds MCM3 (a component of CMG replicative helicase) and promotes chromatin opening. B: in SLNF11-negative cells, ATR alone exerts its replication effects by transiently blocking fork elongation and by activating CHEK1, which also blocks forks and origin firing (not shown). C: at later time, SLFN11 bound to chromatin produces a persistent replication block that irreversibly arrest cell cycle and kills cells. D: in SLFN11-negative cells, replication block by the ATR-CHEK1 is transient and replication can resume after a while.

Figure 5.

Reactivation SLFN11 through ETS transcription factors, viral infection, interferon (IFN) and epigenetic reprogramming. Three types of epigenetic inhibitors (DNA methylation inhibitors, inhibitors for EZH2 [a histone H3K27 methyltransferase] and class I Histone Deacetylase (HDAC) inhibitors) are reported to re-activate SLFN11 in epigenetically inactivated SLFN11-negative cells (see text for details and references).

Figure 6.

Nuclear staining of SLFN11 in colon cancer patient samples. A. SLFN11-positive tumor. B. SLFN11-negative tumor. Note the nuclear staining of SLFN11 in the stroma cells of both samples. Lymphocytes generally have high SLFN11 expression. Paraffin-embedded samples were stained with SLFN11 antibody (D-2): sc-515071X (200 μg/100 μl), mouse monoclonal, Santa Cruz.