RECQL4 alterations in gliomas and nerve sheath tumors: Expression patterns and therapeutic implications

Abstract

RECQL4 plays an important role in maintaining the integrity of the genome and regulating DNA replication. However, the role of RECQL4 in CNS tumors remains unknown. Sequencing data were reviewed and immunohistochemistry was performed on a variety of glial and nerve sheath tumors. Functional studies were performed in glioma (U251) and malignant peripheral nerve sheath tumors (MPNSTs) (NF90-8, ST88-14) cell lines following RECQL4 knockdown and treatment with ATR-inhibitors. Across 1580 CNS tumors, RECQL4 gene variants were identified in 71 cases (4.5%), with 21 (29.6%) of probable pathogenic significance. RECQL4 expression differed significantly across glioma subgroups (P = 0.012). Low-grade gliomas (diffuse: median H-score 57.5; circumscribed: median 130) showed lower expression than high-grade gliomas (median 145, P < 0.05). Neurofibromas displayed higher RECQL4 expression (median 160) compared with MPNSTs (median 97.5, P < 0.001). Among MPNSTs, NF1-associated cases (n = 24, median 95) expressed significantly less RECQL4 than sporadic cases (n = 8, median 162.5, P < 0.001). RECQL4 knockdown in glioma and MPNST cell lines resulted in increased apoptosis and susceptibility to ATR-inhibitors. Our findings show that RECQL4 expression has divergent patterns across tumor types and that targeting RECQL4 may dampen tumor survival and enhance susceptibility to ATR inhibitor therapy in CNS tumors.

Keywords: ATR inhibitors; RecQ helicases; alternative lengthening of telomeres; gliomas; neurofibromatosis type 1.

Figure 1.

RECQL4 immunohistochemistry. Representative microphotographs of RECQL4 immunohistochemistry staining. (A) HGG with high RECQL4 expression (H-score: 180). (B) HGG with intermediate RECQL4 expression (H-score: 80). (C) Angiocentric glioma with low RECQL4 expression (H-score: 10). HGG, adult-type diffuse high-grade glioma.

Figure 2.

RECQL4 immunohistochemistry in gliomas. (A) RECQL4 immunohistochemical H-scores in low-grade circumscribed glioma (LGCG, n = 42), low-grade diffuse glioma (LGDG, n = 4), and high-grade glioma (HGG, n = 65); expression differed significantly across the groups (Kruskal–Wallis test, χ² = 8.78, df = 2, P = 0.012). (B) RECQL4 immunohistochemical H-scores in low-grade circumscribed glioma subtypes, namely subependymal giant-cell astrocytoma (SEGA, n = 2), pilocytic astrocytoma (PA, n = 7), and pleomorphic xanthoastrocytoma (PXA, n = 8). H-scores were similar across groups (Kruskal–Wallis test, χ² = 0.003, df = 2, P = 0.999).

Figure 3.

RECQL4 immunohistochemistry in nerve sheath tumors. RECQL4 H-scores across cases of neurofibromas (n = 67), NF1-related malignant peripheral nerve sheath tumors (MPNST, n = 24), and sporadic MPNST (n = 8). Global comparison performed using Kruskal–Wallis test (χ² = 20.54, df = 2, P = 0.000035).

Figure 4.

RECQL4 knockdown in glioma. Glioma cell line U251 with its RECQL4-knockdowns (RECQL4sh2 and RECQL4sh5) and empty vector control (pLKO.1). Cell numbers measured at multiple time points (upper). Percentage of BrdU-positive cells (lower left). Percentage of apoptotic cells (lower right).

Figure 5.

RECQL4 knockdown in MPNST. Malignant peripheral nerve sheath tumor cell line NF90-8 with its RECQL4-knockdowns (RECQL4sh2 and RECQL4sh5) and empty vector control (pLKO.1). Cell numbers measured at multiple time points (upper). Percentage of BrdU-positive cells (lower left). Percentage of apoptotic cells (lower right).

Figure 6.

RECQL4 knockdown in MPNST. Malignant peripheral nerve sheath tumor cell line ST88-14 with its RECQL4-knockouts (RECQL4sh2 and RECQL4sh5) and empty vector control (pLKO.1). Cell numbers measured at multiple time points (upper). Percentage of BrdU-positive cells (lower left). Percentage of apoptotic cells (lower right).

Figure 7.

ATR inhibition decreases cell growth in tumor cells with RECQL4 loss. Effect of ATR kinase inhibition on cell proliferation of glioma and malignant peripheral nerve sheath tumor (MPNST) cell lines and their RECQL4-knockouts. Glioma U251 cell line survival fraction at different dosages of AZD6738 (Upper left) and VE-821 (Upper right). MPNST NF90-8 cell line survival fraction at different dosages of AZD6738 (Mid left)) and VE-821 (Mid right). MPNST ST88-14 cell line survival fraction at different dosages of AZD6738 (Lower left) and VE-821 (Lower right)).