Marco de Bruyn

POLE Proofreading Mutations Elicit an Antitumor Immune Response in Endometrial Cancer

Purpose: Recent studies have shown that 7% to 12% of endometrial cancers are ultramutated due to somatic mutation in the proofreading exonuclease domain of the DNA replicase POLE. Interestingly, these tumors have an excellent prognosis. In view of the emerging data linking mutation burden, immune response, and clinical outcome in cancer, we investigated whether POLE-mutant endometrial cancers showed evidence of increased immunogenicity. Experimental Design: We examined immune infiltration and activation according to tumor POLE proofreading mutation in a molecularly defined endometrial cancer cohort including 47 POLE-mutant tumors. We sought to confirm our results by analysis of RNAseq data from the TCGA endometrial cancer series and used the same series to examine whether differences in immune infiltration could be explained by an enrichment of immunogenic neoepitopes in POLE-mutant endometrial cancers. Results: Compared with other endometrial cancers, POLE mutants displayed an enhanced cytotoxic T-cell response, evidenced by increased numbers of CD8+ tumor-infiltrating lymphocytes and CD8A expression, enrichment for a tumor-infiltrating T-cell gene signature, and strong upregulation of the T-cell cytotoxic differentiation and effector markers T-bet, Eomes, IFNG, PRF, and granzyme B. This was accompanied by upregulation of T-cell exhaustion markers, consistent with chronic antigen exposure. In silico analysis confirmed that POLE-mutant cancers are predicted to display more antigenic neoepitopes than other endometrial cancers, providing a potential explanation for our findings. Conclusions: Ultramutated POLE proofreading-mutant endometrial cancers are characterized by a robust intratumoral T-cell response, which correlates with, and may be caused by an enrichment of antigenic neopeptides. Our study provides a plausible mechanism for the excellent prognosis of these cancers. Clin Cancer Res; 21(14); 3347–55. ©2015 AACR.

Therapeutic potential of Galectin-9 in human disease

In recent years, an important role has emerged for the glycan‐binding protein Galectin‐9 (Gal‐9) in health and disease. In normal physiology, Gal‐9 seems to be a pivotal modulator of T‐cell immunity by inducing apoptosis in specific T‐cell subpopulations. Because these T‐cell populations are associated with autoimmunity, inflammatory disease, and graft rejection, it was postulated that application of exogenous Gal‐9 may limit pathogenic T‐cell activity. Indeed, treatment with recombinant Gal‐9 ameliorates disease activity in various preclinical models of autoimmunity and allograft graft rejection. In many solid cancers, the loss of Gal‐9 expression is closely associated with metastatic progression. In line with this observation, treatment with recombinant Gal‐9 prevents metastatic spread in various preclinical cancer models. In addition, various hematological malignancies are sensitive to apoptotic elimination by recombinant Gal‐9. Here, we review the biology and physiological role of this versatile lectin and discuss the therapeutic potential of Gal‐9 in various diseases, including autoimmunity, asthma, infection, and cancer.

Carbon monoxide-Releasing Molecule-2 (CORM-2) attenuates acute hepatic ischemia reperfusion injury in rats

BackgroundHepatic ischemia-reperfusion injury (I/Ri) is a serious complication occurring during liver surgery that may lead to liver failure. Hepatic I/Ri induces formation of reactive oxygen species, hepatocyte apoptosis, and release of pro-inflammatory cytokines, which together causes liver damage and organ dysfunction. A potential strategy to alleviate hepatic I/Ri is to exploit the potent anti-inflammatory and cytoprotective effects of carbon monoxide (CO) by application of so-called CO-releasing molecules (CORMs). Here, we assessed whether CO released from CORM-2 protects against hepatic I/Ri in a rat model.MethodsForty male Wistar rats were randomly assigned into four groups (n = 10). Sham group underwent a sham operation and received saline. I/R group underwent hepatic I/R procedure by partial clamping of portal structures to the left and median lobes with a microvascular clip for 60 minutes, yielding ~70% hepatic ischemia and subsequently received saline. CORM-2 group underwent the same procedure and received 8 mg/kg of CORM-2 at time of reperfusion. iCORM-2 group underwent the same procedure and received iCORM-2 (8 mg/kg), which does not release CO. Therapeutic effects of CORM-2 on hepatic I/Ri was assessed by measuring serum damage markers AST and ALT, liver histology score, TUNEL-scoring of apoptotic cells, NFkB-activity in nuclear liver extracts, serum levels of pro-inflammatory cytokines TNF-α and IL-6, and hepatic neutrophil infiltration.ResultsA single systemic infusion with CORM-2 protected the liver from I/Ri as evidenced by a reduction in serum AST/ALT levels and an improved liver histology score. Treatment with CORM-2 also up-regulated expression of the anti-apoptotic protein Bcl-2, down-regulated caspase-3 activation, and significantly reduced the levels of apoptosis after I/Ri. Furthermore, treatment with CORM-2 significantly inhibited the activity of the pro-inflammatory transcription factor NF-κB as measured in nuclear extracts of liver homogenates. Moreover, CORM-2 treatment resulted in reduced serum levels of pro-inflammatory cytokines TNF-α and IL-6 and down-regulation of the adhesion molecule ICAM-1 in the endothelial cells of liver. In line with these findings, CORM-2 treatment reduced the accumulation of neutrophils in the liver upon I/Ri. Similar treatment with an inactive variant of CORM-2 (iCORM-2) did not have any beneficial effect on the extent of liver I/Ri.ConclusionsCORM-2 treatment at the time of reperfusion had several distinct beneficial effects on severity of hepatic I/Ri that may be of therapeutic value for the prevention of tissue damage as a result of I/Ri during hepatic surgery.

Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowth in vitro and in vivo

BackgroundAdvanced melanoma is characterized by a pronounced resistance to therapy leading to a limited patient survival of ~6 - 9 months. Here, we report on a novel bifunctional therapeutic fusion protein, designated anti-MCSP:TRAIL, that is comprised of a melanoma-associated chondroitin sulfate proteoglycan (MCSP)-specific antibody fragment (scFv) fused to soluble human TRAIL. MCSP is a well-established target for melanoma immunotherapy and has recently been shown to provide important tumorigenic signals to melanoma cells. TRAIL is a highly promising tumoricidal cytokine with no or minimal toxicity towards normal cells. Anti-MCSP:TRAIL was designed to 1. selectively accrete at the cell surface of MCSP-positive melanoma cells and inhibit MCSP tumorigenic signaling and 2. activate apoptotic TRAIL-signaling.ResultsTreatment of a panel of MCSP-positive melanoma cell lines with anti-MCSP:TRAIL induced TRAIL-mediated apoptotic cell death within 16 h. Of note, treatment with anti-MCSP:sTRAIL was also characterized by a rapid dephosphorylation of key proteins, such as FAK, implicated in MCSP-mediated malignant behavior. Importantly, anti-MCSP:TRAIL treatment already inhibited anchorage-independent growth by 50% at low picomolar concentrations, whereas > 100 fold higher concentrations of non-targeted TRAIL failed to reduce colony formation. Daily i.v. treatment with a low dose of anti-MCSP:TRAIL (0.14 mg/kg) resulted in a significant growth retardation of established A375 M xenografts. Anti-MCSP:TRAIL activity was further synergized by co-treatment with rimcazole, a σ-ligand currently in clinical trials for the treatment of various cancers.ConclusionsAnti-MCSP:TRAIL has promising pre-clinical anti-melanoma activity that appears to result from combined inhibition of tumorigenic MCSP-signaling and concordant activation of TRAIL-apoptotic signaling. Anti-MCSP:TRAIL alone, or in combination with rimcazole, may be of potential value for the treatment of malignant melanoma.

Somatic POLE proofreading domain mutation, immune response, and prognosis in colorectal cancer: a retrospective, pooled biomarker study

BACKGROUND Precision cancer medicine depends on defining distinct tumour subgroups using biomarkers that may occur at very modest frequencies. One such subgroup comprises patients with exceptionally mutated (ultramutated) cancers caused by mutations that impair DNA polymerase epsilon (POLE) proofreading. METHODS We examined the association of POLE proofreading domain mutation with clinicopathological variables and immune response in colorectal cancers from clinical trials (VICTOR, QUASAR2, and PETACC-3) and colorectal cancer cohorts (Leiden University Medical Centre 1 and 2, Oslo 1 and 2, Bern, AMC-AJCC-II, and Epicolon-1). We subsequently investigated its association with prognosis in stage II/III colorectal cancer by Cox regression of pooled individual patient data from more than 4500 cases from these studies. FINDINGS Pathogenic somatic POLE mutations were detected in 66 (1·0%) of 6517 colorectal cancers, and were mutually exclusive with mismatch repair deficiency (MMR-D) in the 6277 cases for whom both markers were determined (none of 66 vs 833 [13·4%] of 6211; p<0·0001). Compared with cases with wild-type POLE, cases with POLE mutations were younger at diagnosis (median 54·5 years vs 67·2 years; p<0·0001), were more frequently male (50 [75·8%] of 66 vs 3577 [55·5%] of 6445; p=0·0010), more frequently had right-sided tumour location (44 [68·8%] of 64 vs 2463 [39·8%] of 6193; p<0·0001), and were diagnosed at an earlier disease stage (p=0·006, χ2 test for trend). Compared with mismatch repair proficient (MMR-P) POLE wild-type tumours, POLE-mutant colorectal cancers displayed increased CD8+ lymphocyte infiltration and expression of cytotoxic T-cell markers and effector cytokines, similar in extent to that observed in immunogenic MMR-D cancers. Both POLE mutation and MMR-D were associated with significantly reduced risk of recurrence compared with MMR-P colorectal cancers in multivariable analysis (HR 0·34 [95% CI 0·11-0·76]; p=0·0060 and 0·72 [0·60-0·87]; p=0·00035), although the difference between the groups was not significant. INTERPRETATION POLE proofreading domain mutations identify a subset of immunogenic colorectal cancers with excellent prognosis. This association underscores the importance of rare biomarkers in precision cancer medicine, but also raises important questions about how to identify and implement them in practice. FUNDING Cancer Research UK, Academy of Medical Sciences, Health Foundation, EU, ERC, NIHR, Wellcome Trust, Dutch Cancer Society, Dutch Digestive Foundation.

Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects.

IntroductionRheumatoid arthritis (RA) is considered a T cell driven autoimmune disease, therefore, the ability of B cell depleting biologics, e.g., anti-CD20 antibodies, to alleviate RA is unclear. This study examined the proportions of IL-17-secreting lymphocytes in the blood of healthy subjects and RA patients and determined if Th17 cells belong to a CD20+ subset of T cells.MethodsFluorescence-activated cell sorting and confocal microscopy verified CD3, CD4/CD8 and CD20-staining of T cells. IL-17 secretion was determined using a commercial assay.ResultsIn healthy subjects and RA patients blood, the median percentage of total CD20+ lymphocytes was similar (7.5%; n = 6 and 10.3%; n = 9, respectively) and comprised predominantly of B cells (~ 86%). However, 2-4% of CD3+ T cells from both healthy subjects (n = 7) and RA (n = 8) individuals co-expressed CD20. The peripheral blood of healthy subjects contained few IL-17-secreting CD20+ T cells (< 0.1%; n = 6). In contrast, in RA blood a median and interquartile range % of, 24.2%; IQR 28.5 of IL-17-secreting T cells were CD20+ (n = 9; p = 0.02).ConclusionsIn the blood of RA patients, a greater proportion of Th17 cells are of a CD20+ phenotype compared to healthy individuals. These cells may represent an additional target for anti-CD20 therapies.

Potent Systemic Anticancer Activity of Adenovirally Expressed EGFR-Selective TRAIL Fusion Protein

Previously, we demonstrated potent tumor cell-selective pro-apoptotic activity of scFv425:sTRAIL, a recombinant fusion protein comprised of EGFR-directed antibody fragment (scFv425) genetically fused to human soluble TNF-related apoptosis-inducing ligand (sTRAIL). Here, we report on the promising therapeutic systemic tumoricidal activity of scFv425:sTRAIL when produced by the replication-deficient adenovirus Ad-scFv425:sTRAIL. In vitro treatment of EGFR-positive tumor cells with Ad-scFv425:sTRAIL resulted in the potent induction of apoptosis of not only infected tumor cells, but importantly also of up to 60% of noninfected EGFR-positive tumor cells. A single intraocular injection of Ad-scFv425:sTRAIL in tumor-free nu/nu mice resulted in predominant liver infection and concomitant high blood plasma levels of scFv425:sTRAIL. These mice showed no sign of Ad-scFv425:sTRAIL-related liver toxicity. Identical treatment of mice with established intraperitoneal renal cell carcinoma xenografts resulted in rapid and massive tumor load reduction and subsequent long-term survival. Taken together, adenoviral-mediated in vivo production of scFv425:sTRAIL may be exploitable for systemic treatment of EGFR-positive cancer.

Targeted delivery of a designed sTRAIL mutant results in superior apoptotic activity towards EGFR-positive tumor cells

Previously, we have shown that epidermal growth factor receptor (EGFR)-selective delivery of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), by genetic fusion to antibody fragment scFv425, enhances the tumor-selective pro-apoptotic activity of sTRAIL. Insight into the respective contribution of the agonistic receptors TRAIL-R1 and TRAIL-R2 to TRAIL-induced apoptosis may provide a rational approach to further optimize TRAIL-based therapy. Recently, this issue has been investigated using sTRAIL mutants designed to selectively bind to either receptor. However, the relative contribution of the respective TRAIL receptors, in particular TRAIL-R1, in TRAIL signaling is still unresolved. Here, we fused scFv425 to designed sTRAIL mutant sTRAILmR1–5, reported to selectively activate TRAIL-R1, and investigated the therapeutic apoptotic activity of this novel fusion protein. EGFR-specific binding of scFv425:sTRAILmR1–5 potently induced apoptosis, which was superior to the apoptotic activity of scFv425:sTRAIL-wt and a nontargeted MOCK-scFv:sTRAILmR1–5. During cotreatment with cisplatin or the histone deacetylase inhibitor valproic acid, scFv425:sTRAILmR1–5 retained its superior pro-apoptotic activity compared to scFv425:sTRAIL-wt. However, in catching-type Enzyme-Linked ImmunoSorbent Assays with TRAIL-R1:Fc and TRAIL-R2:Fc, scFv425:sTRAILmR1–5 was found to not only bind to TRAIL-R1 but also to TRAIL-R2. Binding to TRAIL-R2 also had functional consequences because the apoptotic activity of scFv425:sTRAILmR1–5 was strongly inhibited by a TRAIL-R2 blocking monoclonal antibody. Moreover, scFv425:sTRAILmR1–5 retained apoptotic activity upon selective knockdown of TRAIL-R1 using small inhibitory RNA. Collectively, these data indicate that both agonistic TRAIL receptors are functionally involved in TRAIL signaling by scFv425:sTRAILmR1–5 in solid tumor cells. Moreover, the superior target cell-restricted apoptotic activity of scFv425:sTRAILmR1–5 indicates its therapeutic potential for EGFR-positive solid tumors.

Cell surface delivery of TRAIL strongly augments the tumoricidal activity of T-cells

Purpose: Adoptive T-cell therapy generally fails to induce meaningful anticancer responses in patients with solid tumors. Here, we present a novel strategy designed to selectively enhance the tumoricidal activity of T cells by targeted delivery of TNF-related apoptosis-inducing ligand (TRAIL) to the T-cell surface. Experimental Design: We constructed two recombinant fusion proteins, anti-CD3:TRAIL and K12:TRAIL. Tumoricidal activity of T cells in the presence of these fusion proteins was assessed in solid tumor cell lines, primary patient-derived malignant cells, and in a murine xenograft model. Results: When added to T cells, K12:TRAIL and anti-CD3:TRAIL selectively bind to the T-cell surface antigens CD3 and CD7, respectively, leading to cell surface accretion of TRAIL. Subsequently, anti-CD3:TRAIL and K12:TRAIL increased the tumoricidal activity of T cells toward cancer cell lines and primary patient-derived malignant cells by more than 500-fold. Furthermore, T-cell surface delivery of TRAIL strongly inhibited tumor growth and increased survival time of xenografted mice more than 6-fold. Conclusions: Targeted delivery of TRAIL to cell surface antigens of T cells potently enhances the tumoricidal activity of T cells. This approach may be generally applicable to enhance the efficacy of adoptive T-cell therapy. Clin Cancer Res; 17(17); 5626–37. ©2011 AACR.

The ever-expanding immunomodulatory role of calreticulin in cancer immunity.

Calreticulin is a pleiotropic molecule that normally resides in the lumen of the endoplasmic reticulum (ER). Here, it has various functions, ranging from regulation of calcium homeostasis to ensuring proper protein folding. More recently, calreticulin gained special interest for its extracellular functions, where it has direct immunomodulatory activity. In this respect, calreticulin activates dendritic cells and macrophages. In addition, certain anti-cancer therapies induce the translocation of calreticulin from the ER to the cell surface of dying cancer cells, where calreticulin dictates the immunogenicity of these cells. Interestingly, treatment with tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) also induces membrane calreticulin exposure on cancer cells. As shown here, calreticulin directly interacts with TRAIL and its receptor-signaling complex, as well as with other TNF family members. Of note, TRAIL is a well known immunomodulatory molecule, and is expressed on the surface of natural killer T-cells. Therefore, calreticulin may have an as yet unrecognized wide(r) impact on immunity, with the TNF-ligand family modulating virtually all aspects of the immune response.