Germline variants of DNA repair and immune genes in lymphoma from lymphoma-cancer families.

The genetic predisposition to lymphoma is not fully understood. We identified 13 lymphoma-cancer families (2011-2021), in which 27 individuals developed lymphomas and 26 individuals had cancers. Notably, male is the predominant gender in lymphoma patients, whereas female is the predominant gender in cancer patients (p = .019; OR = 4.72, 95% CI, 1.30-14.33). We collected samples from 18 lymphoma patients, and detected germline variants through exome sequencing. We found that germline protein truncating variants (PTVs) were enriched in DNA repair and immune genes. Totally, we identified 31 heterozygous germline mutations (including 12 PTVs) of 25 DNA repair genes and 19 heterozygous germline variants (including 7 PTVs) of 14 immune genes. PTVs of ATM and PNKP were found in two families, respectively. We performed whole genome sequencing of diffuse large B cell lymphomas (DLBCLs), translocations at IGH locus and activation of oncogenes (BCL6 and MYC) were verified, and homologous recombination deficiency was detected. In DLBCLs with germline PTVs of ATM, deletion and insertion in CD58 were further revealed. Thus, in lymphoma-cancer families, we identified germline defects of both DNA repair and immune genes in lymphoma patients.

Limonin inhibits the stemness of cancer stem-like cells derived from colorectal carcinoma cells potentially via blocking STAT3 signaling.

BACKGROUND: Limonin is one of the most abundant active ingredients of Tetradium ruticarpum. It exerts antitumor effects on several kinds of cancer cells. However, whether limonin exerts antitumor effects on colorectal cancer (CRC) cells and cancer stem-like cells (CSCs), a subpopulation responsible for a poor prognosis, is unclear. AIM: To evaluate the effects of limonin on CSCs derived from CRC cells. METHODS: CSCs were collected by culturing CRC cells in serum-free medium. The cytotoxicity of limonin against CSCs and parental cells (PCs) was determined by cholecystokinin octapeptide-8 assay. The effects of limonin on stemness were detected by measuring stemness hallmarks and sphere formation ability. RESULTS: As expected, limonin exerted inhibitory effects on CRC cell behaviors, including cell proliferation, migration, invasion, colony formation and tumor formation in soft agar. A relatively low concentration of limonin decreased the expression stemness hallmarks, including Nanog and ß-catenin, the proportion of aldehyde dehydrogenase 1-positive CSCs, and the sphere formation rate, indicating that limonin inhibits stemness without presenting cytotoxicity. Additionally, limonin treatment inhibited invasion and tumor formation in soft agar and in nude mice. Moreover, limonin treatment significantly inhibited the phosphorylation of STAT3 at Y705 but not S727 and did not affect total STAT3 expression. Inhibition of Nanog and ß-catenin expression and sphere formation by limonin was obviously reversed by pretreatment with 2 µmol/L colievlin. CONCLUSION: Taken together, these results indicate that limonin is a promising compound that targets CSCs and could be used to combat CRC recurrence and metastasis.

Different situations of identifying second primary malignant tumors in lymphoma patients with synchronous solid tumors.

BACKGROUND: To our knowledge, the different situations of identifying second primary malignant tumors (SPMTs) in lymphoma patients with synchronous solid tumors remain to be comprehensively investigated. METHODS: We retrospectively collected information pertaining to lymphoma patients with synchronous solid tumors (diagnosed within 6 months) at Peking University Cancer Hospital & Institute between 2009 and 2019. The non-parametric Aalen-Johansen estimator was applied to calculate cumulative incidence function in the competing risk model. Furthermore, propensity score-matched analysis was performed to compare survival differences in lymphoma patients with or without synchronous solid tumors. RESULTS: Thirty-eight patients were enrolled. There were three situations of identifying SPMTs. First, in 15 patients (39.5%), SPMTs were identified before the initiation of any treatment. Among them, priority was given to anti-lymphoma treatment in case of only three patients. Second, in 17 patients (44.7%), SPMTs were unexpectedly detected on surgical specimen assessment; of them, 13 received anti-lymphoma treatment after surgery. Third, in six patients (15.8%), SPMTs were identified after the outset of treatment for the primary tumor; in this population, three of four patients with lymphoma switched toward the treatment plan for SPMTs. The 5-year overall survival was 58.7%. The cumulative incidence function within 5 years was 26.6% for lymphoma and 14.7% for other solid tumors. The early identification of SPMTs was associated with better outcomes (p = 0.048). After balancing the baseline characteristics, no differences in survival were observed between lymphoma patients with and without synchronous solid tumors (p = 0.664). CONCLUSIONS: This is the first study to present the different situations of identifying SPMTs in lymphoma patients with synchronous solid tumors. In only <50% patients, SPMTs were identifiable at baseline. SPMT identification at different situations may make it difficult to choose the optimal therapeutic option, which may consequently impact patient survival.

The Serum- and Glucocorticoid-Inducible Kinase 1 (SGK1) as a Novel Therapeutic Target in Mantle Cell Lymphoma.

INTRODUCTION: Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell-derived malignant disease. MCL is treated using general chemotherapy; however, disease progression and relapse are common; thus, the development of novel therapeutic targets for treatment of MCL is urgently required. Serum- and glucocorticoid-inducible kinase 1 (SGK1) is involved in various cellular activities, and its dysregulation contributes to the pathogenesis of multiple types of cancer. However, little is known regarding its functional roles and associated molecular mechanisms in MCL. METHODS: SGK1 inhibition mediated by either shRNA or treatment with SGK1 inhibitor (GSK650394) was conducted in MCL cell lines. Western blotting analysis was performed to figure out the expression of related proteins. MCL-cell-derived xenograft models were constructed to evaluate the anti-tumor effects of SGK1 inhibition or/and Bruton's tyrosine kinase (BTK) inhibition in vivo. RESULTS: In this study, it was shown that inhibition of SGK1 significantly reduced cell proliferation, invasion and migration, increased apoptosis and blocked cell cycle progression in MCL cells. Furthermore, SGK1 inhibition significantly reduced the activation of ERK, AKT/mTOR, JAK2/STAT3 and the NF-κB signaling pathways. Using MCL-cell-derived xenograft mice models, SGK1 inhibition decreased tumor cell proliferation and tumor growth. Importantly, SGK1 overexpression significantly promoted xenograft tumor growth. Moreover, simultaneous inhibition of SGK1 and Bruton tyrosine kinase (BTK) resulted in synergistic anti-tumor effects on MCL both in vitro and in vivo. CONCLUSION: SGK1 may be a novel candidate therapeutic target and simultaneous inhibition of SGK1 and BTK may be a promising therapeutic strategy for MCL patients. Further pre-clinical and even clinical studies of SGK1 inhibitor or combination with BTK inhibitor are essential.

Ibrutinib combined with low-dose histone deacetylases inhibitor chidamide synergistically enhances the anti-tumor effect in B-cell lymphoma.

Aberrant activity of histone deacetylases (HDACs) is frequently detected in B-cell lymphomas, which indicated the therapeutic implications of HDAC inhibitors for B-cell malignancies. We have discovered that lymphoma cells treated with HDAC inhibitor presented with activation of Bruton tyrosine kinase (BTK) which played an important role in the development of B-cell malignancies. Therefore, our study intended to explore whether the addition of ibrutinib (BTK inhibitor) to chidamide (HDAC inhibitor) could generate combined anti-tumor effects in B-cell lymphomas. Using cell viability assay, cell cycle and apoptosis kit, we demonstrated an evident synergistic action of ibrutinib and chidamide in inhibiting tumor cell proliferation and motility. Consistent with in vitro data, the synergistic anti-tumor effects were also observed in multiple tumor-bearing mice models. By performing RNA-seq and flow cytometry of tumor tissue, the enhancement of anti-tumor immunity was observed with the co-treatment of chidamide and ibrutinib. Together, these mechanistic insights indicated that simultaneously targeting BTK and HDAC could be a promising clinical therapy for B-cell lymphomas.

Survival and prognostic analysis of T-cell lymphoblastic lymphoma patients treated with dose-adjusted BFM-90 regimen.

We aimed to investigate the long-term prognosis and prognostic factors of T-cell lymphoblastic lymphoma (T-LBL) patients who received dose-adjusted Berlin-Frankfurt-Münster (BFM)-90 regimen as first-line therapy in our center. A total of 145 T-LBL patients who underwent first-line dose-adjusted BFM-90 was retrospectively reviewed. Conditional survival analysis was used to evaluate the long-term prognosis of patients. Receiver operating characteristic (ROC) curve was applied to determine the optimal cut-off value for neutrophil-to-lymphocyte ratio (NLR). Estimated 3-year overall survival (OS) and progression-free survival (PFS) rates for overall were 66.8% and 58.4%, respectively. Conditional survival analysis showed that for patients having survived 3 and 5 years or more after the completion of the treatment, the estimated subsequent 3-year OS thereafter increased to 85.7% and 94.3, respectively. Patients receiving consolidation APBSCT (Autologous peripheral blood stem cell transplantation) after BFM-90 regimen had superior 3-year OS than those with non-APBSCT (79.1% vs. 33.4%, p<0.001). We also discovered that baseline NLR ≥4.95 was negatively associated with OS (HR=2.75, 95% CI 1.55-4.89, p=0.015) and PFS (HR=2.07, 95% CI 1.25-4.96, p=0.021) via multivariable analysis. Conclusions: The survival probability of T-LBL patients treated with first-line dose-adjusted BFM-90 has improved significantly as patients have survived for every additional year. The addition of consolidation APBSCT following dose-adjusted BFM-90 therapy bring further survival benefits for those patients. Baseline NLR ≥4.95 was an independent risk factor for T-LBL patients in our study.

Addition of BTK inhibitor orelabrutinib to rituximab improved anti-tumor effects in B cell lymphoma.

Bruton tyrosine kinase (BTK) inhibitor ibrutinib has been validated as an effective drug to treat B cell malignancies. Combined therapies comprising ibrutinib and anti-CD20 antibodies like rituximab were designed as a backbone in many clinical trials. However, the off-target inhibition of ibrutinib on interleukin-2 (IL-2)-inducible T cell kinase (ITK) may reduce rituximab's antibody-dependent cellular cytotoxicity (ADCC) efficacy. Orelabrutinib (Orel), a novel BTK inhibitor, was designed with high selectivity to BTK. In our study, we demonstrated in preclinical models that orelabrutinib in combination with rituximab could preserve NK-cell-mediated ADCC induced by rituximab and enhanced the apoptosis of tumor cells in vitro. The addition of orelabrutinib to rituximab had produced promising combined anti-tumor effects in B cell lymphomas in vivo. Collectively, combination therapy of orelabrutinib with rituximab would benefit patients with B cell lymphoma, especially those with relapsed or refractory disease.

Germline variants of DNA repair genes in early onset mantle cell lymphoma.

Although somatic mutations of DNA repair genes are frequent in mantle cell lymphoma (MCL), our understanding of their germline defects is limited. In a Chinese family with maternal Lynch syndrome and paternal B cell non-Hodgkin lymphoma, one sibling developed both Lynch syndrome and MCL. Lynch syndrome is caused by heterozygous mutations in mismatch repair (MMR) genes. To understand the genetic predispositions in the family, we performed exome sequencing and analyses of affected individuals and their tumor samples. A novel germline indel, MLH1 Gly101fsX1, was identified as the cause of Lynch syndrome, and unstable microsatellite loci and mutational signatures as evidence of defective MMR were revealed in the MCL sample. Furthermore, we included additional 15 MCL patients with early onset, and found by exome sequencing that 11 patients carried heterozygous germline variants of 20 DNA repair genes, including MSH2 in MMR. In the MCL with MSH2 Arg359fsX16, unstable microsatellite loci and defective MMR signatures were also found. In addition, five patients also had heterozygous germline variants of genes involved in B cell functions. Thus, our study found germline variants of genes in single-strand break repair, double-strand break repair, and Fanconi anemia pathway in early onset MCL; and for the first time we identified germline defects of MMR in two MCLs.

ITK inhibition induced in vitro and in vivo anti-tumor activity through downregulating TCR signaling pathway in malignant T cell lymphoma.

BACKGROUND: Angioimmunoblastic T cell lymphoma (AITL) is a distinct subtype of peripheral T cell lymphoma and associated with poor outcomes. The activation status of T cell receptor (TCR) signaling has recently become a focus of attention in terms of the therapeutic targets. However, the molecular pathogenesis mechanisms and novel therapeutic targets are largely unknown. METHODS: Antibodies specific to phosphorylated ZAP70, ITK and PLCγ1 were used to identify the activation status of intracellular proteins involved in TCR signaling in AITL patients. Malignant T cell lymphoma cells were transduced with a lentiviral construct containing ITK shRNA for cellular and functional assays. The antitumor effects of the selective ITK inhibitor BMS-509744 were determined in vitro and in vivo. RESULTS: Immunohistochemistry staining showed that more than half of the AITL patients (n = 38) exhibited continuously activated intracellular TCR signaling pathway. Patients positive for phosphorylated ITK showed a lower rate of complete response (20% vs. 75%, P = 0.004) and a shorter progression-free survival (5.17 months vs. 25.1 months, P = 0.022) than patients negative for phosphorylated ITK. Genetic and pharmacological cellular ITK inhibition significantly compromised the proliferation, invasion and migration of malignant T cells. The selective ITK inhibitor BMS-509744 also induced the pro-apoptotic effects and G2/M phase cell cycle arrest in vitro and in vivo. Finally, inhibition of ITK synergistically enhanced the antitumor effect of vincristine and doxorubicin on malignant T cell lymphoma cell lines. CONCLUSIONS: Our findings suggest that ITK may be a novel candidate therapeutic target for the treatment of patients with ITK-expressing malignant T-cell lymphomas.

Combination of Enzastaurin and Ibrutinib synergistically induces anti-tumor effects in diffuse large B cell lymphoma.

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma. Although durable remissions can be achieved in more than half of these patients, DLBCL remains a significant clinical challenge, with approximately 30% of patients not being cured. BCR-associated kinases (SYK, BTK, and PI3K) inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers. Early studies demonstrated that protein kinase C-ß (PKCß) inhibitors alter phosphorylation level the Bruton's tyrosine kinase (BTK), which leads to enhanced BTK signaling. Here, for the first time, we investigate whether the combination of PKCß inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL. METHODS: In vitro cell proliferation was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay. Induction of apoptosis and cell cycle arrest were measured by flow cytometry. Western Blotting analysis was used to detect the essential regulatory enzymes in related signaling pathways. RNA-seq was conducted to evaluate the whole transcriptome changes brought by co-treatment with low doses of enzastaurin and ibrutinib. The synergistic anti-tumor effects of enzastaurin and ibrutinib were also evaluated in vivo. RESULTS: Combination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of DLBCL cells, including reduction of proliferation, promoting apoptosis, inducting G1 phase arrest, preventing cell invasion and migration, and down-regulating activation of downstream signaling. More importantly, whole-transcriptome changes results showed that combination therapy worked synergistically to regulate whole-transcriptome expression compared with enzastaurin and ibrutinib alone. Co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathway. Furthermore, the mRNA expression analysis further indicated that co-treatment significantly decreased the mRNA levels of NOTCH1. The combination effect in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment also was demonstrated in vivo. CONCLUSIONS: Combination of enzastaurin and ibrutinib had synergistic anti-tumor effects in DLBCL, independent of molecular subtype. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCß might be a new treatment strategy for DLBCL.

The proto-oncogene Mer tyrosine kinase is a novel therapeutic target in mantle cell lymphoma.

BACKGROUND: Mantle cell lymphoma (MCL) is an incurable B cell-derived malignant tumor with a median overall survival of 4-5 years. Mer tyrosine kinase (MerTK) has been reported to be aberrantly expressed in leukemia, melanoma, and gastric cancer, and plays a pivotal role in the process of oncogenesis. This study assessed the role of MerTK in MCL for the first time. METHODS: Immunohistochemistry and western blot were performed to figure out expression of MerTK in MCL. MerTK inhibition by either shRNA or treatment with UNC2250 (a MerTK-selective small molecular inhibitor) was conducted in MCL cell lines. MCL-cell-derived xenograft models were established to evaluate the anti-tumor effects of UNC2250 in vivo. RESULTS: MerTK was ectopically expressed in four of six MCL cell lines. Sixty-five of 132 (48.9%) MCL patients showed positive expression of MerTK. MerTK inhibition by either shRNA or treatment with UNC2250 decreased activation of downstream AKT and p38, inhibited proliferation and invasion in MCL cells, and sensitized MCL cells to treatment with vincristine in vitro and doxorubicin in vitro and in vivo. UNC2250 induced G2/M phase arrest and prompted apoptosis in MCL cells, accompanied by increased expression of Bax, cleaved caspase 3 and poly (ADP-ribose) polymerase, and decreased expression of Bcl-2, Mcl-1 and Bcl-xL. Moreover, UNC2250 delayed disease progression in MCL-cell-derived xenograft models. CONCLUSIONS: Our data prove that ectopic MerTK may be a novel therapeutic target in MCL, and further pre-clinical or even clinical studies of UNC2250 or new MerTK inhibitors are essential and of great significance.

Prognostic role of serum miRNA-16 in primary gastric lymphoma.

Primary gastric lymphoma (PGL) is the most common extranodal non-Hodgkin lymphoma. However, uniform criteria for treatment and outcome evaluation are lacking. MicroRNA-16 (miRNA-16) has been shown to exhibit tumor-suppressive properties in many cancers and is a good biomarker of prognosis. We detected the expression level of miRNA-16 in 91 patients with PGL by qPCR and analyzed the prognostic significance. The proportion of patients with high expression of miRNA-16 was greater in patients with disease onset at age ≤ 60 years (P = 0.027), early-stage disease (P = 0.06), and marginal zone B-cell lymphoma (P = 0.0031). Furthermore, the proportion of patients with high expression of miRNA-16 was greater in those with a complete response than in those without a complete response (P = 0.054). However, miRNA-16 expression was not a statistically significant predictor of survival. These results suggest that miRNA-16 is a useful biomarker of disease prognosis in patients with PGL.

The mTOR kinase inhibitor everolimus synergistically enhances the anti-tumor effect of the Bruton's tyrosine kinase (BTK) inhibitor PLS-123 on Mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive and incurable malignant disease. Despite of general chemotherapy, relapse and mortality are common, highlighting the need for the development of novel targeted drugs or combination of therapeutic regimens. Recently, several drugs that target the B-cell receptor (BCR) signaling pathway, especially the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, have demonstrated notable therapeutic effects in relapsed/refractory patients, which indicate that pharmacological inhibition of BCR pathway holds promise in MCL treatment. Here, we have developed a novel irreversible BTK inhibitor, PLS-123, that has more potent and selective anti-tumor activity than ibrutinib in vitro and in vivo. Using in vitro screening, we discovered that the combination of PLS-123 and the mammalian target of rapamycin (mTOR) inhibitor everolimus exert synergistic activity in attenuating proliferation and motility of MCL cell lines. Simultaneous inhibition of BTK and mTOR resulted in marked induction of apoptosis and cell cycle arrest in the G1 phase, which were accompanied by upregulation of pro-apoptotic proteins (cleaved Caspase-3, cleaved PARP and Bax), repression of anti-apoptotic proteins (Mcl-1, Bcl-xl and XIAP), and downregulation of regulators of the G1/S phase transition (CDK2, CDK4, CDK6 and Cyclin D1). Gene expression profile analysis revealed simultaneous treatment with these agents led to inhibition of the JAK2/STAT3, AKT/mTOR signaling pathways and SGK1 expression. Finally, the anti-tumor and pro-apoptotic activities of combination strategy have also been demonstrated using xenograft mice models. Taken together, simultaneous suppression of BTK and mTOR may be indicated as a potential therapeutic modality for the treatment of MCL.

TP53 Arg72 as a favorable prognostic factor for Chinese diffuse large B-cell lymphoma patients treated with CHOP.

BACKGROUND: TP53 Arg72Pro (SNP rs1042522) is associated with risk of non-Hodgkin lymphoma (NHL). Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL. However, the relationship between this SNP and prognosis of DLBCL in Asians is unknown. METHODS: Genotyping of TP53 Arg72Pro was done in 425 Chinese DLBCL patients. Two hundred and eighty-nine patients were treated with R-CHOP, and 136 patients received CHOP or CHOP-like as frontline regimen. Three hundred and ninety-six patients were assessable for the efficacy. RESULTS: Patients with Arg/Arg and Arg/Pro at codon 72 of TP53 had a higher complete response rate (61% vs. 44%, P = 0.007) than those with Pro/Pro. In the subgroup treated with CHOP or CHOP-like therapy, patients with Arg/Arg and Arg/Pro showed a higher 5-year overall survival (OS) rate than those with Pro/Pro (68.8% vs. 23.2%, P = 0.001). Multivariate Cox regression analysis revealed TP53 Arg72 as a favorable prognostic factor in this group. However, the combination of rituximab with CHOP significantly increased the 5-year OS rate of patients with Pro/Pro to 63%. CONCLUSION: This study revealed TP53 Arg72 as a favorable prognostic factor for Chinese DLBCL patients treated with CHOP or CHOP-like as frontline therapy.

EP300 single nucleotide polymorphism rs20551 correlates with prolonged overall survival in diffuse large B cell lymphoma patients treated with R-CHOP.

BACKGROUND: Rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) is used as standard frontline regimen for diffuse large B-cell lymphoma (DLBCL). The landscape of somatic mutations in DLBCL revealed that inactivation of EP300 plays an important role in lymphomagenesis. A common EP300 single nucleotide polymorphism (SNP) rs20551 results in the substitution of valine for isoleucine at codon 997 close to the Bromodomain. However, the association between SNP rs20551 and clinical prognosis in DLBCL patients treated with R-CHOP is unknown. METHODS: In this study we analyzed the EP300 SNP rs20551 and prognosis of 226 DLBCL patients who treated with R-CHOP or R-CHOP-like regimes from 2002 to 2013. Determination of the EP300 SNP rs20551 from genomic DNA was obtained by Sanger chain termination sequencing. RESULT: In this study, the frequency of the A and G allele of the EP300 SNP rs20551 in 226 patients were 92.5 and 7.5%, respectively. We did not observe obvious correlation between patients' disease features and the EP300 SNP rs20551. But the patients with genotype AA had a higher 5-year overall survival rate than those with genotype GA (77.0% vs. 64.7%, p = 0.045). Furthermore, multivariate Cox regression analysis showed that the GA genotype of EP300 SNP rs20551 was an independent poor prognostic factor for DLBCL patients treated with Rituximab-chemotherapy (p = 0.009, HR 2.956, 95% CI 1.315-6.645). CONCLUSION: This study suggests that EP300 SNP rs20551 might be a useful biomarker to predict the long-term outcome of R-CHOP in DLBCL patients.

CD275-Independent IL-17-Producing T Follicular Helper-like Cells in Lymphopenic Autoimmune-Prone Mice.

T cells undergo homeostatic expansion and acquire an activated phenotype in lymphopenic microenvironments. Restoration of normal lymphocyte numbers typically re-establishes normal homeostasis, and proinflammatory cytokine production returns to baseline. Mice deficient in guanine nucleotide exchange factor RasGRP1 exhibit dysregulated homeostatic expansion, which manifests as lymphoproliferative disease with autoantibody production. Our previous work revealed that autoreactive B cells lacking RasGRP1 break tolerance early during development, as well as during germinal center responses, suggesting that T cell-independent and T cell-dependent mechanisms are responsible. Examination of whether a particular T cell subset is involved in the breach of B cell tolerance revealed increased Th17 cells in Rasgrp1-deficient mice relative to control mice. Rasgrp1-deficient mice lacking IL-17R had fewer germinal centers, and germinal centers that formed contained fewer autoreactive B cells, suggesting that IL-17 signaling is required for a break in B cell tolerance in germinal centers. Interestingly, a fraction of Th17 cells from Rasgrp1-deficient mice were CXCR5(+) and upregulated levels of CD278 coordinate with their appearance in germinal centers, all attributes of T follicular helper cells (Tfh17). To determine whether CD278-CD275 interactions were required for the development of Tfh17 cells and for autoantibody, Rasgrp1-deficient mice were crossed with CD275-deficient mice. Surprisingly, mice deficient in RasGRP1 and CD275 formed Tfh17 cells and germinal centers and produced similar titers of autoantibodies as mice deficient in only RasGRP1. Therefore, these studies suggest that requirements for Tfh cell development change in lymphopenia-associated autoimmune settings.

Analysis of genetic variants of the poly(ADP-ribose) polymerase-1 gene in breast cancer in French patients.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that catalyzes the poly(ADP-ribosyl)ation of target proteins in response to DNA damage and has been proposed to play a role in DNA repair, recombination, transcription, cell death, cell proliferation, as well as in stabilization of the genome. We have recently shown that PARP-1 deficiency causes mammary tumorigenesis in mice. In the present study, we investigated whether genetic variants and single nucleotide polymorphisms (SNPs) of PARP-1 contribute to human breast cancer. To this end, we screened all PARP-1 exons, 7.1kb of intron-exon junction and 1.0-kb promoter sequences in 83 French patients with breast cancer and 100 controls by direct sequencing of genomic DNA. Twenty rare genetic variants of PARP-1, including c.1148C>A (Ser383Tyr), c.1354C>A (Arg452Arg), c.2819A>G (Lys940Arg) were detected in nine (10.8%) breast cancers of these patients. Among 31 polymorphic sites examined, five haplotype-tagging SNPs (htSNPs) of PARP-1 were identified. Interestingly, the genotype distribution of htSNP c.852T>C (Ala284Ala) was likely associated with loss of estrogen- and progesterone-receptor expression. The present study implies that genetic variants of PARP-1 may contribute to breast cancerogenesis and that PARP-1 htSNP c.852T>C (Ala284Ala) may influence hormonal therapy of breast cancer.

PARP1 Val762Ala polymorphism reduces enzymatic activity.

Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K(m) of PARP1-Ala762 was increased to a 1.2-fold of the K(m) of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K(m). This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding the cancer risk of carriers of the PARP1 Val762Ala polymorphism.