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TOSO/FAIM3 has recently been identified as the long sought after Fc receptor for IgM (FcµR). FcµR is expressed on human CD19+ B cells, CD4+/CD8+ T cells, and CD56+/CD3− NK cells, and has been shown to be overexpressed in chronic lymphocytic leukemia (CLL) cells. CLL is a malignancy of mature IgM+ B lymphocytes that display features of polyreactive, partially anergized B cells related to memory B cells. Herein, we report that FcµR is O-glycosylated in its extracellular domain and identify the major sites of O-glycosylation. By employing immunofluorescence confocal microscopy, we found that FcµR localized to the cell membrane, but also found that large pools of FcµR accumulate in the trans-Golgi network. Aggregation of FcµR on CLL cells by IgM prompted rapid internalization of both IgM and FcµR, reaching half maximal internalization of cell bound IgM within one minute. Upon internalization, FcµR transported IgM through the endocytic pathway to the lysosome, where it was degraded. Using a series of FcµR deletion mutants, we identified a proline-rich domain essential for cell surface expression of FcµR and a second domain, containing a YXXΦ motif, that controls internalization. While it has been reported, that BCR activation increases FcµR expression; we found that activation of TLRs strongly downregulated FcµR at both the mRNA and protein levels. Through internalization of IgM bound immune complexes, FcµR may play a role in immune surveillance and contribute to B-cell activation. In addition, FcµR deserves study as a potential pathway for the delivery of therapeutic antibody-drug conjugates into CLL cells.

The online version of this article contains a supplementary appendix. BackgroundIn chronic lymphocytic leukemia lenalidomide causes striking immune activation, possibly leading to clearance of tumor cells. We conducted this study to investigate the mechanism of action of lenalidomide and the basis for its unique toxicities in chronic lymphocytic leukemia. Design and MethodsPatients with relapsed chronic lymphocytic leukemia were treated with lenalidomide 20 mg (n=10) or 10 mg (n=8) daily for 3 weeks on a 6-week cycle. Correlative studies assessed expression of co-stimulatory molecules on tumor cells, T-cell activation, cytokine levels, and changes in lymphocyte subsets. ResultsLenalidomide upregulated the co-stimulatory molecule CD80 on chronic lymphocytic leukemia and mantle cell lymphoma cells but not on normal peripheral blood B cells in vitro. T-cell activation was apparent in chronic lymphocytic leukemia, weak in mantle cell lymphoma, but absent in normal peripheral blood mononuclear cells and correlated with the upregulation of CD80 on B cells. Strong CD80 upregulation and T-cell activation predicted more severe side effects, manifesting in 83% of patients as a cytokine release syndrome within 8-72 h after the first dose of lenalidomide. Serum levels of various cytokines, including tumor necrosis factor-α, increased during treatment. CD80 upregulation on tumor cells correlated with rapid clearance of leukemic cells from the peripheral blood. In contrast, neither the severity of the cytokine release syndrome nor the degree of T-cell activation in vitro correlated with clinical response. ConclusionsUpregulation of CD80 on tumor cells and T-cell activation correlate with unique toxicities of lenalidomide in chronic lymphocytic leukemia. However, T-cell activation appears to be dispensable for the drug's anti-tumor effects. This provides a rationale for combinations of lenalidomide with fludarabine or alemtuzumab.

Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct β-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses. Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed and, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling. We show that anti-progastrin antibodies decrease self-renewal of CSCs both and, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors. Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS-mutated colorectal patients, for which there is a crucial unmet medical need. .

A pilot study previously demonstrated that thrice-weekly, fractionated-dose intravenous rituximab (RTX) limits CD20 loss from chronic lymphocytic leukemia (CLL) B cells, thereby enhancing immunotherapeutic targeting. Here, we investigated the feasibility of giving 20 mg rituximab subcutaneously thrice weekly for up to 12 weeks in 4 previously treated CLL patients. Subcutaneous rituximab was well-tolerated with minimal injection site reactions; a variable degree of efficacy was observed, likely influenced by the size of the patients' B cell/CD20 burden. Subcutaneous RTX largely preserved CD20 expression on leukemic cells but the most effective therapeutic dosing regimen needs to be established (ClinicalTrials.gov Identifier: NCT00366418). Haematologica. 2010;95:329-332. doi: 10.3324/haematol.2009 This is an open-access paper. IntroductionThe anti-CD20 monoclonal antibody rituximab (RTX) has shown remarkable efficacy in non-Hodgkin's lymphomas (NHL).1,2 However, compared to therapy for NHL, RTX therapy in chronic lymphocytic leukemia (CLL) is associated with lower response rates.3 Possible explanations include lower CD20 levels on CLL cells compared to NHL cells. Alternatively, due to high tumor burden or substantial leukemic disease, there can be exhaustion of effector mechanisms which kill RTX-targeted CLL B cells, such as antibodydependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).4,5 Another potential limitation for standard dose, single-agent intravenous RTX therapy in CLL, manifested after saturation/exhaustion of clearance mechanisms, is the shaving reaction in which RTX/CD20 immune complexes on B cells are removed by effector cells expressing FcγR. 5,6 This process can reduce or completely abrogate the efficacy of subsequent RTX dosing. Fractionated dosing schedules that limit exhaustion of effector mechanisms may be more effective than current intravenous bolus schedules of 375 mg/m 2 RTX. A pilot trial suggested that low-dose RTX at 20 mg/m 2 intravenously thrice weekly promotes clearance of leukemic cells without inducing substantial loss of targeted CD20.7 This trial was limited to a four-week treatment duration and required frequent clinic visits for patients. In principle, low RTX doses can be selfadministered subcutaneously. If subcutaneous administration is safe and effective, it could be more convenient for patients than intravenous treatment and would make fractionated dosing over prolonged periods possible. Based on ease of use and tolerability considerations, subcutaneous injections in this pilot study were limited to 2 mL/day, thrice weekly, allowing for RTX doses of 20 mg due to its fixed formulation at 10 mg/mL. Design and Methods PatientsThis pilot phase I study used 20 mg subcutaneous RTX doses thrice weekly for 6-12 weeks (ClinicalTrials.gov Identifier: NCT00366418). Criteria for inclusion were active CLL, previous fludarabine treatment, CD20 expression on leukemic

Background: In colorectal cancer, hPG80 (progastrin) is released from tumor cells, promotes cancer stem cells (CSC) self-renewal and is detected in the blood of patients. Because the gene GAST that encodes hPG80 is a target gene of oncogenic pathways that are activated in many tumor types, we hypothesized that hPG80 could be expressed by tumors from various origins other than colorectal cancers, be a drug target and be detectable in the blood of these patients. Methods: hPG80 expression was monitored by fluorescent immunohistochemistry and mRNA expression in tumors from various origins. Cancer cell lines were used in sphere forming assay to analyze CSC self-renewal. Blood samples were obtained from 1546 patients with 11 different cancer origins and from two retrospective kinetic studies in patients with peritoneal carcinomatosis or hepatocellular carcinomas. These patients were regularly sampled during treatments and assayed for hPG80. Findings: We showed that hPG80 was present in the 11 tumor types tested. In cell lines originating from these tumor types, hPG80 neutralization decreased significantly CSC self-renewal by 28 to 54%. hPG80 was detected in the blood of patients at significantly higher concentration than in healthy blood donors (median hPG80: 4.88 pM versus 1.05 pM; p < 0.0001) and shown to be correlated to GAST mRNA levels in the matched tumor (i.e., lung cancers, Spearman r = 0.8; p = 0.0023). Furthermore, we showed a strong association between longitudinal hPG80 concentration changes and anti-cancer treatment efficacy in two independent retrospective studies. In the peritoneal carcinomatosis cohort, median hPG80 from inclusion to the post-operative period decreased from

Precise management of kidney cancer requires the identification of prognostic factors. hPG80 (circulating progastrin) is a tumor promoting peptide present in the blood of patients with various cancers, including renal cell carcinoma (RCC). In this study, we evaluated the prognostic value of plasma hPG80 in 143 prospectively collected patients with metastatic RCC (mRCC). The prognostic impact of hPG80 levels on overall survival (OS) in mRCC patients after controlling for hPG80 levels in non-cancer age matched controls was determined and compared to the International Metastatic Database Consortium (IMDC) risk model (good, intermediate, poor). ROC curves were used to evaluate the diagnostic accuracy of hPG80 using the area under the curve (AUC). Our results showed that plasma hPG80 was detected in 94% of mRCC patients. hPG80 levels displayed high predictive accuracy with an AUC of 0.93 and 0.84 when compared to 18–25 year old controls and 50–80 year old controls, respectively. mRCC patients with high hPG80 levels (>4.5 pM) had significantly lower OS compared to patients with low hPG80 levels (<4.5 pM) (12 versus 31.2 months, respectively; p = 0.0031). Adding hPG80 levels (score of 1 for patients having hPG80 levels > 4.5 pM) to the six variables of the IMDC risk model showed a greater and significant difference in OS between the newly defined good-, intermediate- and poor-risk groups (p = 0.0003 compared to p = 0.0076). Finally, when patients with IMDC intermediate-risk group were further divided into two groups based on hPG80 levels within these subgroups, increased OS were observed in patients with low hPG80 levels (<4.5 pM). In conclusion, our data suggest that hPG80 could be used for prognosticating survival in mRCC alone or integrated to the IMDC score (by adding a variable to the IMDC score or by substratifying the IMDC risk groups), be a prognostic biomarker in mRCC patients.

Histone deacetylase inhibitors (HDACi) have demonstrated promising therapeutic potential in clinical trials for hematological malignancies. HDACi, such as SAHA/Vorinostat, Trichostatin A, and MS-275 were found to induce apoptosis of leukemic blasts through activation of the death receptor pathway and transcriptional induction of the Tumor Necrosis Factor (TNF)-related pro-apoptotic family members, TRAIL and FasL. The impact of HDACi on TNF-related costimulatory molecules such as 4-1BB ligand (4-1BBL/TNFSF9) is however not known. Following exposure to SAHA/Vorinostat, Trichostatin A, and MS-275, transcript levels were determined by real time PCR in Jurkat, Raji and U937 cells. Treatment with HDACi up-regulated TNFSF9 gene expression in the three leukemia cell lines, yet to different extend and with distinct kinetics, which did not require de novo protein synthesis and was not associated with DNAse I hypersensitive chromatin remodeling. Transcriptional activity of TNFSF9 promoter-luciferase constructs was induced up to 12 fold by HDACi, and implication of Sp1/Sp3 transcription factors binding to functional GC-box elements was evidenced by reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays. Functionality of modulated target genes was assessed in allogeneic mixed leukocyte reaction experiments. MS-275- and to a lesser extent Trichostatin A- and SAHA-treated Raji cells significantly up regulated T lymphocytes proliferation which was reduced by about 50% by a 4-1BB blocking recombinant protein, while MS-275- but neither Trichostatin A- nor SAHA-treated cells up-regulated IFNγ secretion by T lymphocytes. Our results identify 4-1BBL/4-1BB as a downstream target of HDACi, especially of MS-275 anti-leukemia action in vitro. Thus, HDACi such as MS-275 displaying dual TNF-dependent proapoptotic and costimulatory activities might be favored for inclusion in HDACi-based anti-cancer therapeutic strategies.