SAMHD1 Mutations and Expression in Mantle Cell Lymphoma Patients

Abstract

SAMHD1 (sterile alpha motif domain and histidine-aspartate domain-containing protein 1) is a deoxynucleoside triphosphate triphosphohydrolase regulating innate immune and modulating DNA damage signaling. It plays an important role in the development of some tumors. SAMHD1 was also reported as a barrier to cytarabine, a common chemotherapy drug for mantle cell lymphoma (MCL), and as a biomarker of grim prognosis for acute myelocytic leukemia (AML) patients. However, SAMHD1 expression and function in MCL have not been well-defined. In the present study, we evaluated SAMHD1 expression by immunohistochemistry and its gene structure by Sanger sequencing in MCL. Our results showed that SAMHD1 was positive in 36 (62.1%) patients. Importantly, SAMHD1-positive patients were associated with lower chemotherapy response rate (p = 0.023) and shorter overall survival (p = 0.039) than SAMHD1-negative cases. These results suggest that SAMHD1 is an adverse biomarker for MCL patients, which is due to the high expression of SAMHD1 and rapid cell proliferation. These findings were confirmed in an in vitro study using the siRNA technique. Silencing the SAMHD1 gene in the MCL cell line Jeko-1 significantly decreased cell proliferation and increased cell apoptosis. The MCL cell line with SAMHD1 knockdown showed lower Ki-67 proliferation index, higher caspase-3, and higher sensitivity to cytarabine. Furthermore, for the first time, four previously unreported missense mutations (S302Y, Y432C, E449G, and R451H) in exon 8 and exon 12 of the SAMHD1 gene were discovered by sequencing. The mutations had not been found to corelate with SAMHD1 protein expression detected by immunohistochemistry. The biological functions of this mutated SAMHD1 remain to be investigated.

Keywords: SAMHD1; cytarabine resistance; gene silencing; immunohistochemistry; mantle cell lymphoma; mutations; patient risk stratification; prognosis.

Figure 1

Representation of IHC and FISH results of mantle cell lymphoma (MCL) tissues. (A) Morphology of MCL. Tumor cells were small to medium and were diffusely arranged in the mantle and paracortex area of the lymph node (HE ×400). Panels (B–D) are the results of IHC staining (×400) for cyclin D1 (B), SOX11 (C), and SAMHD1 in MCL tissues (D) and the insert in (D) is SAMHD1-positive control from clear cell kidney carcinoma. Panels (E–G) (×1,000) are examples of positive FISH tests of t(11;14) IGH/CCND1 translocation and cytokine gene rearrangement in MCL tissues. Panel (E) shows positive for t(14;18) IGH/CCND1 translocation and panel (G) is positive for CCND1 gene rearrangement. CCND2 gene amplification was also detected in one case of MCL (F). Inserts in panels (E–G) are their corresponding negative controls.

Figure 2

Selected sequencing chromatograms of SAMHD1 using Sanger sequencing. Panel (A) shows the homogeneous c905 C>A mutation (red box highlighted) in exon 8 of SAMHD1. Panel (B) shows the heterogeneous c1295A>G transition in exon 12 of SAMHD1. In panel (C), the highlighted red box represents the homogeneous c1346G>A transition in exon 12 of SAMHD1. Panel (D) is the heterogeneous c1552G>A mutation in exon 12 of SAMHD1. The included table on the right is the correlation of SAM HD1 mutations and their expression in MCL cases.

Figure 3

SAMHD1 promotes the proliferation, inhibits the apoptosis, and mediates the resistance to cytarabine of MCL cells. MCL cells (Jeko-1) were cultured and their SAMHD1 gene was silenced by using two lentiviral vectors SAMHD1-shRNA1 and 2 to generate two SAMHD1-silenced Jeko-1 cell lines (shSAMHD1-1 and shSAMHD1-2). The expression of SAMHD1 in wild-type Jeko-1 cells and two SAMHD1-silenced Jeko-1 cell lines detected by Western blot (A). The proliferation of Jeko-1 cells. Wild-type Jeko-1 cells showed higher proliferation activity than two SAMHD1-silenced cell lines, shSAMHD1-1 and shSAMHD1-2 (B). Panel (C) is the Western blot of Ki-67 expression. Wild-type Jeko-1 cells expressed a higher level of Ki-67 protein than the two silenced cell lines, shSAMHD1-1 and shSAMHD1-2. SAMHD1-silenced Jeko-1 cells (both shSAMHD1-1 and shSAMHD1-2) had a higher proportion of apoptosis cells than the wild-type parent cell (D). (E) The expression of caspase-3 in the SAMHD1-1 silenced cells was the same as in wild-type Jeko-1 cells, while the level of cleaved-caspase-3 in the silenced cells was higher than the wild-type cells (E). IC50 of cytarabine for shSAMHD1-1 and shSAMHD1-2 cells was lower than that for wild-type Jeko-1 (F), indicating that SAMHD1 promoted cytarabine resistance. The symbol "*" represents p < 0.5, and "***" represents p < 0.001.

Figure 4

Relationship of the SAMHD1 expression with clinical outcome of MCL patients. There was no significance in PFS between SAMHD1-positive and SAMHD1-negative patients (A). However, SAMHD1-positive patients had shorter OS than SAMHD1-negative patients (B). Among low- to intermediate-risk MCL patients (MIPI score between 0 and 5), SAMHD1-positive patients had worse PFS (C) and OS (D) than SAMHD1-negative patients.