Stromal cell inhibition of anti-CD20 antibody mediated killing of B-cell malignancies.

Introduction: The glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab has been licensed for treatment in follicular non-Hodgkin lymphoma and B-CLL following clinical trials demonstrating superior outcomes to standard of care treatment. However, ultimately many patients still relapse, highlighting the need to understand the mechanisms behind treatment failure to improve patient care. Resistance to chemotherapy is often caused by the ability of malignant B-cells to migrate to the bone marrow and home into the stromal layer. Therefore, this study aimed to investigate whether stromal cells were also able to inhibit type II anti-CD20 antibody mechanisms of action, contributing to resistance to therapy. Methods: A stromal-tumor co-culture was established in vitro between Raji or Daudi B-cell tumor cells and M210B4 stromal cells in 24 well plates. Results: Contact with stromal cells was able to protect tumor cells from obinutuzumab mediated programmed cell death (PCD), antibody dependent cellular phagocytosis and antibody dependent cellular cytotoxicity. Furthermore, such protection required direct contact between stroma and tumor cells. Stromal cells appeared to interfere with obinutuzumab mediated B-cell homotypic adhesion through inhibiting and reversing actin remodelling, potentially as a result of stromal-tumor cell contact leading to downregulation of CD20 on the surface of tumor cells. Further evidence for the potential role of CD20 downregulation comes through the reduction in surface CD20 expression and inhibition of obinutuzumab mediated PCD when tumor cells are treated with Ibrutinib in the presence of stromal cells. The proteomic analysis of tumor cells after contact with stromal cells led to the identification of a number of altered pathways including those involved in cell adhesion and the actin cytoskeleton and remodeling. Discussion: This work demonstrates that contact between tumor cells and stromal cells leads to inhibition of Obinutuzumab effector functions and has important implications for future therapies to improve outcomes to anti-CD20 antibodies. A deeper understanding of how anti-CD20 antibodies interact with stromal cells could prove a useful tool to define better strategies to target the micro-environment and ultimately improve patient outcomes in B-cell malignancies.

Multi-platform profiling characterizes molecular subgroups and resistance networks in chronic lymphocytic leukemia.

Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.

Rituximab and obinutuzumab differentially hijack the B cell receptor and NOTCH1 signaling pathways.

The anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B cell death. To characterize the signaling processes responsible for improved B cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B cell receptor. Although both antibodies induce strong ERK and MYC activation sufficient to promote cell-cycle arrest and B cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, by impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments.

Natural NS3 resistance polymorphisms occur frequently prior to treatment in HIV-positive patients with acute hepatitis C.

NS3 protease inhibitors are set to improve sustained virological response rates in HIV-positive patients with hepatitis C. We measured the prevalence of natural resistance polymorphisms in 38 acutely infected treatment-naive patients using direct and deep sequencing. Twenty six percent of patients (10/38) had a majority variant resistance mutation (in order of frequency; Q80K - 16%, V36M - 5%, T54S - 3%, V55A - 3%, and D168A - 3%). Low-frequency mutations were detected in all samples. Further studies are required to determine threshold levels associated with treatment failure.

Human papillomavirus gene expression is controlled by host cell splicing factors.

HPVs (human papillomaviruses) infect stratified epithelia and cause a variety of lesions ranging from benign warts to invasive tumours. The virus life cycle is tightly linked to differentiation of the keratinocyte it infects: papillomaviruses modulate host gene expression to ensure efficient virus replication. For example, the viral transcription factor E2 can directly up-regulate, in an epithelial differentiation-dependent manner, cellular SRSFs [SR (serine/arginine-rich) splicing factors] that control constitutive and alternative splicing. Changes in alternative splicing and the mechanisms controlling this for viral mRNAs have been the subject of intense exploration. However, to date experiments have only been carried out in model systems because the genetic systems suitable for studying alternative splicing of viral RNAs in the context of the virus life cycle are relatively recent and technically challenging. Now using these life cycle-supporting systems, our laboratory has identified SR proteins as important players in differentiation-dependent regulation of HPV gene expression. Better understanding of the role of cellular factors in regulating the virus life cycle is needed as it may help development of novel diagnostic approaches and antiviral therapies in the future.

The novel Bcl-2 inhibitor ABT-737 is more effective in hypoxia and is able to reverse hypoxia-induced drug resistance in neuroblastoma cells.

Neuroblastoma is a common solid tumor of childhood and advanced disease carries a poor prognosis despite intensive multimodality therapy. Hypoxia is a common feature of solid tumors because of poorly organized tumor-induced neovasculature. Hypoxia is associated with advanced stage and poor outcome in a range of tumor types, and leads to resistance to clinically relevant cytotoxic agents in neuroblastoma and other pediatric tumors in vitro. Resistance to apoptosis is a common feature of tumor cells and leads to pleiotropic drug resistance, mediated by Bcl-2 family proteins. ABT-737 is a novel small-molecule inhibitor of Bcl-2 and Bcl-x(L) that is able to induce apoptosis in a range of tumor types. Neuroblastoma cell lines are relatively resistant to ABT-737-induced apoptosis in normoxia, but in contrast to the situation with conventional cytotoxic agents are more sensitive in hypoxia. This sensitization is because of an increase in ABT-737-induced apoptosis and is variably dependent upon the presence of functional hypoxia-inducible factor 1 (HIF-1) α. In contrast to the situation in colon carcinoma and non-small cell lung cancer cells, hypoxia does not result in downregulation of the known ABT-737 resistance factor, Mcl-1, nor any other Bcl-2 family proteins. ABT-737 sensitizes neuroblastoma cells to clinically relevant cytotoxic agents under normal levels of oxygen, and importantly, this sensitization is maintained under hypoxia when neuroblastoma cells are resistant to these agents. Thus rational combinations of ABT-737 and conventional cytotoxics offer a novel approach to overcoming hypoxia-induced drug resistance in neuroblastoma.

Structure of an atypical Tudor domain in the Drosophila Polycomblike protein.

Post-translational modifications of histone tails are among the most prominent epigenetic marks and play a critical role in transcriptional control at the level of chromatin. The Polycomblike (Pcl) protein is part of a histone methyltransferase complex (Pcl-PRC2) responsible for high levels of histone H3 K27 trimethylation. Studies in Drosophila larvae suggest that Pcl is required for anchoring Pcl-PRC2 at target genes, but how this is achieved is unknown. Pcl comprises a Tudor domain and two PHD fingers. These domains are known to recognize methylated lysine or arginine residues and could contribute to targeting of Pcl-PRC2. Here, we report an NMR structure of the Tudor domain from Drosophila Pcl (Pcl-Tudor) and binding studies with putative ligands. Pcl-Tudor contains an atypical, incomplete aromatic cage that does not interact with known Tudor domain ligands, such as methylated lysines or arginines. Interestingly, human Pcl orthologs exhibit a complete aromatic cage, suggesting that they may recognize methylated lysines. Structural comparison with other Tudor domains suggests that Pcl-Tudor may engage in intra- or intermolecular interactions through an exposed hydrophobic surface patch.

Up-regulation of the embryonic self-renewal network through reversible polyploidy in irradiated p53-mutant tumour cells.

We have previously documented that transient polyploidy is a potential cell survival strategy underlying the clonogenic re-growth of tumour cells after genotoxic treatment. In an attempt to better define this mechanism, we recently documented the key role of meiotic genes in regulating the DNA repair and return of the endopolyploid tumour cells (ETC) to diploidy through reduction divisions after irradiation. Here, we studied the role of the pluripotency and self-renewal stem cell genes NANOG, OCT4 and SOX2 in this polyploidy-dependent survival mechanism. In irradiation-resistant p53-mutated lymphoma cell-lines (Namalwa and WI-L2-NS) but not sensitive p53 wild-type counterparts (TK6), low background expression of OCT4 and NANOG was up-regulated by ionising radiation with protein accumulation evident in ETC as detected by OCT4/DNA flow cytometry and immunofluorescence (IF). IF analysis also showed that the ETC generate PML bodies that appear to concentrate OCT4, NANOG and SOX2 proteins, which extend into complex nuclear networks. These polyploid tumour cells resist apoptosis, overcome cellular senescence and undergo bi- and multi-polar divisions transmitting the up-regulated OCT4, NANOG and SOX2 self-renewal cassette to their descendents. Altogether, our observations indicate that irradiation-induced ETC up-regulate key components of germ-line cells, which potentially facilitate survival and propagation of the tumour cell population.

Generation of monoclonal antibody targeting fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and pathological cells and tissues.

Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells.

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcgammaR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DR-specific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion-related cell death occurs through a lysosome-dependent pathway.

Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes.

PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. Here we report the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state.

A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities.

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pho is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, we purified Pho protein complexes from Drosophila embryos and found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product dSfmbt. Analysis of PhoRC reveals that dSfmbt is a novel PcG protein that is essential for HOX gene repression in Drosophila. PhoRC is bound at HOX gene PREs in vivo, and this targeting strictly depends on Pho-binding sites. Characterization of dSfmbt protein shows that its MBT repeats have unique discriminatory binding activity for methylated lysine residues in histones H3 and H4; the MBT repeats bind mono- and di-methylated H3-K9 and H4-K20 but fail to interact with these residues if they are unmodified or tri-methylated. Our results establish PhoRC as a novel Drosophila PcG protein complex that combines DNA-targeting activity (Pho) with a unique modified histone-binding activity (dSfmbt). We propose that PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state.

The histone methyltransferases Trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins.

Transcriptional on and off states of HOX genes and other developmental control genes are maintained by antagonistic regulators encoded by trithorax group (trxG) and Polycomb group (PcG) genes. The trxG proteins Ash1 and hTRX and the PcG repressor E(z) are histone methyltransferases (HMTases) that methylate distinct lysine residues in the N-terminal tail of histone H3. trxG proteins are generally thought to function as activators of HOX genes, but how histone methylation by Ash1 and Trx promotes HOX gene transcription is not clear. Here, we show that in ash1 and trx mutants expression of HOX genes is lost within their normal expression domains, but we find that, contrary to expectation, this expression is restored in ash1 and trx mutants that also lack PcG gene function. Moreover, such trxG PcG double mutants show severe misexpression of HOX genes and, hence, ectopic activation of HOX genes caused by the removal of PcG gene function also occurs in the absence of ash1 and trx function. Together, these results suggest that the Ash1 and Trx HMTases are not "coactivators" required for transcriptional activation of HOX genes, but function specifically as anti-repressors. We propose that histone methylation by Ash1 and Trx is required continuously throughout development to prevent inappropriate PcG silencing of HOX genes in cells in which they must stay transcriptionally active.