DRE-1/FBXO11, A Conserved F Box Protein, Regulates Apoptosis in C. elegans and is Mutated in Human Lymphoma

In the course of metazoan embryonic and post-embryonic development, more cells are generated than exist in the mature organism, and these cells are deleted by the process of programmed cell death. In addition, cells can be pushed toward death when they accumulate genetic errors, are virally-infected or are otherwise deemed potentially-harmful to the overall organism. Caenorhabditis elegans has proved to be an excellent model system for elucidating the genetic underpinnings of cell death, and research has shown that the core machinery, made up of the egl-1, ced-9, ced-4 and ced-3 genes, is conserved across metazoans, and their homologues are crucial for such diseases as cancer, neurodegeneration and autoimmunity. We used the C. elegans tail-spike cell as a model to uncover dre-1/FBXO11 as a conserved apoptotic regulator that controls tail-spike cell death and also plays a role in human lymphoma. The tail-spike cell is unique among cells fated for programmed cell death in two ways. First, unlike most dying C. elegans cells, the tail-spike cell lives for several hours before its demise, and during this time differentiates. In comparison, most cells die minutes after birth as undifferentiated cells. Second, while tail-spike cell death requires both the CED-3 caspase and CED-4/Apaf-1 adaptor proteins, the BH3-only protein EGL-1 is dispensable. Thus, other gene(s) substitute for egl-1\u27s role as a regulator of caspase activation in this cell, and we set out to identify the relevant gene or genes. A screen for mutants in which the tail-spike cell survives inappropriately yielded a mutant, ns39, in which the ced-3 caspase is transcribed as normal, but fails to become activated. We mapped and cloned this mutant, and found that dre-1 (daf-12-redundant-1) is required for caspase activation in the tail-spike cell. Expression of the dre-1 cDNA in the tail-spike cell rescues the dre-1(ns39) defect in a cell autonomous manner, and expression studies show that dre-1 is expressed in the tail-spike cell. Partial loss-of-function alleles of dre-1, when combined with weak loss-of-function alleles of ced-3 and ced-4, a null allele of egl-1, or a weak gain-of-function allele of ced-9 exhibit a synergistic loss of tailspike cell death. A null allele of ced-9, however, when combined with a strong loss-of-function dre-1 allele, suppresses the tail-spike cell death phenotype of the dre-1(ns39). This epistatic relationship shows that dre-1 acts upstream of, or in parallel to ced-9. These results show that dre-1 has a central role in tail-spike cell death, and are consistent with it acting in place of egl-1 to promote tail-spike cell death. DRE-1 is an F box protein, and we showed via RNAi and genetic experiments that DRE-1 acts in an SCF complex with CUL-1 and SKR-1 to regulate tail-spike cell death, and present evidence that DRE-1 and CED-9 bind to each other in vitro. These results suggest a model in which the dre-1 SCF complex ubiquitinates CED-9 to eliminate its anti-apoptotic function and open the way for the cell to die. A collaboration with Louis Staudt and Lixin Rui at the National Cancer Institute revealed that dre-1ʼs human homologue, FBXO11 is mutated or deleted in 5% of germinal center-like diffuse large B cell lymphomas, and that reintroduction of the gene into lymphoma cell lines that have deleted it induces apoptosis. In addition, FBXO11 binds to BCL2 in lymphoma cell lines and induces the degradation of BCL2, and expression of BCL2 rescues the toxicity of FBXO11. Taken together, our results establish dre-1/FBXO11 as a regulator of apoptosis in C. elegans and human lymphoma, and suggest a model in which DRE-1/FBXO11 ubiquitinates and degrades BCL2, a major anti-apoptotic protein

A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220+CD5+ Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo, we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment

BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient\u27s hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual\u27s TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing

Internal standard-based analysis of microarray data2—Analysis of functional associations between HVE-genes

In this work we apply the Internal Standard-based analytical approach that we described in an earlier communication and here we demonstrate experimental results on functional associations among the hypervariably-expressed genes (HVE-genes). Our working assumption was that those genetic components, which initiate the disease, involve HVE-genes for which the level of expression is undistinguishable among healthy individuals and individuals with pathology. We show that analysis of the functional associations of the HVE-genes is indeed suitable to revealing disease-specific differences. We show also that another possible exploit of HVE-genes for characterization of pathological alterations is by using multivariate classification methods. This in turn offers important clues on naturally occurring dynamic processes in the organism and is further used for dynamic discrimination of groups of compared samples. We conclude that our approach can uncover principally new collective differences that cannot be discerned by individual gene analysi

Transcriptional profiling of macrophages in situ in metastatic melanoma reveals localization-dependent phenotypes and function.

Modulation of immune function at the tumor site could improve patient outcomes. Here, we analyze patient samples of metastatic melanoma, a tumor responsive to T cell-based therapies, and find that tumor-infiltrating T cells are primarily juxtaposed to CD14+ monocytes/macrophages rather than melanoma cells. Using immunofluorescence-guided laser capture microdissection, we analyze transcriptomes of CD3+ T cells, CD14 + monocytes/macrophages, and melanoma cells in non-dissociated tissue. Stromal CD14+ cells display a specific transcriptional signature distinct from CD14+ cells within tumor nests. This signature contains LY75, a gene linked with antigen capture and regulation of tolerance and immunity in dendritic cells (DCs). When applied to TCGA cohorts, this gene set can distinguish patients with significantly prolonged survival in metastatic cutaneous melanoma and other cancers. Thus, the stromal CD14+ cell signature represents a candidate biomarker and suggests that reprogramming of stromal macrophages to acquire DC function may offer a therapeutic opportunity for metastatic cancers

Internal standard-based analysis of microarray data2—Analysis of functional associations between HVE-genes

In this work we apply the Internal Standard-based analytical approach that we described in an earlier communication and here we demonstrate experimental results on functional associations among the hypervariably-expressed genes (HVE-genes). Our working assumption was that those genetic components, which initiate the disease, involve HVE-genes for which the level of expression is undistinguishable among healthy individuals and individuals with pathology. We show that analysis of the functional associations of the HVE-genes is indeed suitable to revealing disease-specific differences. We show also that another possible exploit of HVE-genes for characterization of pathological alterations is by using multivariate classification methods. This in turn offers important clues on naturally occurring dynamic processes in the organism and is further used for dynamic discrimination of groups of compared samples. We conclude that our approach can uncover principally new collective differences that cannot be discerned by individual gene analysis