Highly Multiplexed and Simultaneous Characterization of Protein and RNA in Single Cells by Flow or Mass Cytometry Platforms Using Proximity Ligation Assay for RNA.

In situ hybridization of oligonucleotide probes to intracellular RNA allows quantification of predefined gene transcripts within millions of single cells using cytometry platforms. Previous methods have been hindered by the number of RNA that can be analyzed simultaneously. Here we describe a method called proximity ligation assay for RNA (PLAYR) that permits highly multiplexed RNA analysis that can be combined with antibody staining. Potentially any number of RNA combined with antigen can be analyzed together, being limited only by the number of analytes that can be measured simultaneously.

Soluble CTLA-4 attenuates T cell activation and modulates anti-tumor immunity.

CTLA-4 is a crucial immune checkpoint receptor involved in the maintenance of immune homeostasis, tolerance, and tumor control. Antibodies targeting CTLA-4 have been promising treatments for numerous cancers, but the mechanistic basis of their anti-tumoral immune-boosting effects is poorly understood. Although the ctla4 gene also encodes an alternatively spliced soluble variant (sCTLA-4), preclinical/clinical evaluation of anti-CTLA-4-based immunotherapies have not considered the contribution of this isoform. Here, we explore the functional properties of sCTLA-4 and evaluate the efficacy of isoform-specific anti-sCTLA-4 antibody targeting in a murine cancer model. We show that expression of sCTLA-4 by tumor cells suppresses CD8+ T cells in vitro and accelerates growth and experimental metastasis of murine tumors in vivo. These effects were accompanied by modification of the immune infiltrate, notably restraining CD8+ T cells in a non-cytotoxic state. sCTLA-4 blockade with isoform-specific antibody reversed this restraint, enhancing intratumoral CD8+ T cell activation and cytolytic potential, correlating with therapeutic efficacy and tumor control. This previously unappreciated role of sCTLA-4 suggests that the biology and function of multi-gene products of immune checkpoint receptors need to be fully elucidated for improved mechanistic understanding of cancer immunotherapies.

Roles of PI3Kγ and PI3Kδ in mantle cell lymphoma proliferation and migration contributing to efficacy of the PI3Kγ/δ inhibitor duvelisib.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma that is incurable with existing therapies, and therefore presents a significant unmet clinical need. The ability of this disease to overcome therapy, including those that target the B cell receptor pathway which has a pathogenic role in MCL, highlights the need to develop new treatment strategies. Herein, we demonstrate that a distinguishing feature of lymph node resident MCL cells is the expression of phosphatidylinositol 3-kinase γ (PI3Kγ), a PI3K isoform that is not highly expressed in other B cells or B-cell malignancies. By exploring the role of PI3K in MCL using different PI3K isoform inhibitors, we provide evidence that duvelisib, a dual PI3Kδ/γ inhibitor, has a greater effect than PI3Kδ- and PI3Kγ-selective inhibitors in blocking the proliferation of primary MCL cells and MCL cell lines, and in inhibiting tumour growth in a mouse xenograft model. In addition, we demonstrated that PI3Kδ/γ signalling is critical for migration of primary MCL cells and cell lines. Our data indicates that aberrant expression of PI3Kγ is a critical feature of MCL pathogenesis. Thus, we suggest that the dual PI3Kδ/γ duvelisib would be effective for the treatment of mantle cell lymphoma.

PKCß Facilitates Leukemogenesis in Chronic Lymphocytic Leukaemia by Promoting Constitutive BCR-Mediated Signalling.

B cell antigen receptor (BCR) signalling competence is critical for the pathogenesis of chronic lymphocytic leukaemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKCα function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKCßII upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKCα-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKCα-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKCα-KR expressing cells, coincident with upregulation of PKCßII: supported by activation of key signalling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKCßII inhibitor) treatment of PKCα-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKCßII in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKCα-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKCß expression facilitates leukemogenesis and identify that BCR-mediated signalling is a key driver of CLL development in the PKCα-KR model.

Kinobead Profiling Reveals Reprogramming of BCR Signaling in Response to Therapy within Primary CLL Cells.

PURPOSE: B-cell receptor (BCR) signaling is critical for the pathogenesis of chronic lymphocytic leukemia (CLL), promoting both malignant cell survival and disease progression. Although vital, understanding of the wider signaling network associated with malignant BCR stimulation is poor. This is relevant with respect to potential changes in response to therapy, particularly involving kinase inhibitors. In the current study, we describe a novel high-resolution approach to investigate BCR signaling in primary CLL cells and track the influence of therapy on signaling response. EXPERIMENTAL DESIGN: A kinobead/mass spectrometry-based protocol was used to study BCR signaling in primary CLL cells. Longitudinal analysis of samples donated by clinical trial patients was used to investigate the impact of chemoimmunotherapy and ibrutinib on signaling following surface IgM engagement. Complementary Nanostring and immunoblotting analysis was used to verify our findings. RESULTS: Our protocol isolated a unique, patient-specific signature of over 30 kinases from BCR-stimulated CLL cells. This signature was associated with 13 distinct Kyoto Encyclopedia of Genes and Genomes pathways and showed significant change in cells from treatment-naïve patients compared with those from patients who had previously undergone therapy. This change was validated by longitudinal analysis of clinical trials samples where BCR-induced kinome responses in CLL cells altered between baseline and disease progression in patients failing chemoimmunotherapy and between baseline and treatment in patients taking ibrutinib. CONCLUSIONS: These data comprise the first comprehensive proteomic investigation of the BCR signaling response within CLL cells and reveal unique evidence that these cells undergo adaptive reprogramming of this signaling in response to therapy.

A novel transgenic mouse strain expressing PKCßII demonstrates expansion of B1 and marginal zone B cell populations.

Protein kinase Cß (PKCß) expressed in mammalian cells as two splice variants, PKCßI and PKCßII, functions in the B cell receptor (BCR) signaling pathway and contributes to B cell development. We investigated the relative role of PKCßII in B cells by generating transgenic mice where expression of the transgene is directed to these cells using the Eµ promoter (Eµ-PKCßIItg). Our findings demonstrate that homozygous Eµ-PKCßIItg mice displayed a shift from IgD+IgMdim toward IgDdimIgM+ B cell populations in spleen, peritoneum and peripheral blood. Closer examination of these tissues revealed respective expansion of marginal zone (MZ)-like B cells (IgD+IgM+CD43negCD21+CD24+), increased populations of B-1 cells (B220+IgDdimIgM+CD43+CD24+CD5+), and higher numbers of immature B cells (IgDdimIgMdimCD21neg) at the expense of mature B cells (IgD+IgM+CD21+). Therefore, the overexpression of PKCßII, which is a phenotypic feature of chronic lymphocytic leukaemia cells, can skew B cell development in mice, most likely as a result of a regulatory influence on BCR signaling.

Loss of BAP1 expression is associated with an immunosuppressive microenvironment in uveal melanoma, with implications for immunotherapy development.

Immunotherapy using immune checkpoint inhibitors (ICIs) induces durable responses in many metastatic cancers. Metastatic uveal melanoma (mUM), typically occurring in the liver, is one of the most refractory tumours to ICIs and has dismal outcomes. Monosomy 3 (M3), polysomy 8q, and BAP1 loss in primary uveal melanoma (pUM) are associated with poor prognoses. The presence of tumour-infiltrating lymphocytes (TILs) within pUM and surrounding mUM - and some evidence of clinical responses to adoptive TIL transfer - strongly suggests that UMs are indeed immunogenic despite their low mutational burden. The mechanisms that suppress TILs in pUM and mUM are unknown. We show that BAP1 loss is correlated with upregulation of several genes associated with suppressive immune responses, some of which build an immune suppressive axis, including HLA-DR, CD38, and CD74. Further, single-cell analysis of pUM by mass cytometry confirmed the expression of these and other markers revealing important functions of infiltrating immune cells in UM, most being regulatory CD8+ T lymphocytes and tumour-associated macrophages (TAMs). Transcriptomic analysis of hepatic mUM revealed similar immune profiles to pUM with BAP1 loss, including the expression of IDO1. At the protein level, we observed TAMs and TILs entrapped within peritumoural fibrotic areas surrounding mUM, with increased expression of IDO1, PD-L1, and ß-catenin (CTNNB1), suggesting tumour-driven immune exclusion and hence the immunotherapy resistance. These findings aid the understanding of how the immune response is organised in BAP1 - mUM, which will further enable functional validation of detected biomarkers and the development of focused immunotherapeutic approaches. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Multiplexed profiling of RNA and protein expression signatures in individual cells using flow or mass cytometry.

Advances in single-cell analysis technologies are providing novel insights into phenotypic and functional heterogeneity within seemingly identical cell populations. RNA within single cells can be analyzed using unbiased sequencing protocols or through more targeted approaches using in situ hybridization (ISH). The proximity ligation assay for RNA (PLAYR) approach is a sensitive and high-throughput technique that relies on in situ and proximal ligation to measure at least 27 specific RNAs by flow or mass cytometry. We provide detailed instructions for combining this technique with antibody-based detection of surface/internal protein, allowing simultaneous highly multiplexed profiling of RNA and protein expression at single-cell resolution. PLAYR overcomes limitations on multiplexing seen in previous branching DNA-based RNA detection techniques by integration of a transcript-specific oligonucleotide sequence within a rolling-circle amplification (RCA). This unique transcript-associated sequence can then be detected by heavy metal (for mass cytometry)- or fluorophore (for flow cytometry)-conjugated complementary detection oligonucleotides. Included in this protocol is methodology to label oligonucleotides with lanthanide metals for use in mass cytometry. When analyzed by mass cytometry, up to 40 variables (with scope for future expansion) can be measured simultaneously. We used the described protocol to demonstrate intraclonal heterogeneity within primary cells from chronic lymphocytic leukemia patients, but it can be adapted to other primary cells or cell lines in suspension. This robust, reliable and reproducible protocol can be completed in 2-3 d and can be paused at several stages for convenience.

New Perspectives, Opportunities, and Challenges in Exploring the Human Protein Kinome.

The human protein kinome comprises 535 proteins that, with the exception of approximately 50 pseudokinases, control intracellular signaling networks by catalyzing the phosphorylation of multiple protein substrates. While a major research focus of the last 30 years has been cancer-associated Tyr and Ser/Thr kinases, over 85% of the kinome has been identified to be dysregulated in at least one disease or developmental disorder. Despite this remarkable statistic, for the majority of protein kinases and pseudokinases, there are currently no inhibitors progressing toward the clinic, and in most cases, details of their physiologic and pathologic mechanisms remain at least partially obscure. By curating and annotating data from the literature and major public databases of phosphorylation sites, kinases, and disease associations, we generate an unbiased resource that highlights areas of unmet need within the kinome. We discuss strategies and challenges associated with characterizing catalytic and noncatalytic outputs in cells, and describe successes and new frontiers that will support more comprehensive cancer-targeting and therapeutic evaluation in the future. Cancer Res; 78(1); 15-29. ©2017 AACR.

Lck is a relevant target in chronic lymphocytic leukaemia cells whose expression variance is unrelated to disease outcome.

Pathogenesis of chronic lymphocytic leukaemia (CLL) is contingent upon antigen receptor (BCR) expressed by malignant cells of this disease. Studies on somatic hypermutation of the antigen binding region, receptor expression levels and signal capacity have all linked BCR on CLL cells to disease prognosis. Our previous work showed that the src-family kinase Lck is a targetable mediator of BCR signalling in CLL cells, and that variance in Lck expression associated with ability of BCR to induce signal upon engagement. This latter finding makes Lck similar to ZAP70, another T-cell kinase whose aberrant expression in CLL cells also associates with BCR signalling capacity, but also different because ZAP70 is not easily pharmacologically targetable. Here we describe a robust method of measuring Lck expression in CLL cells using flow cytometry. However, unlike ZAP70 whose expression in CLL cells predicts prognosis, we find Lck expression and disease outcome in CLL are unrelated despite observations that its inhibition produces effects that biologically resemble the egress phenotype taken on by CLL cells treated with idelalisib. Taken together, our findings provide insight into the pathobiology of CLL to suggest a more complex relationship between expression of molecules within the BCR signalling pathway and disease outcome.

Transcriptional mechanism of vascular endothelial growth factor-induced expression of protein kinase CßII in chronic lymphocytic leukaemia cells.

A key feature of chronic lymphocytic leukaemia (CLL) cells is overexpressed protein kinase CßII (PKCßII), an S/T kinase important in the pathogenesis of this and other B cell malignancies. The mechanisms contributing to enhanced transcription of the gene coding for PKCßII, PRKCB, in CLL cells remain poorly described, but could be important because of potential insight into how the phenotype of these cells is regulated. Here, we show that SP1 is the major driver of PKCßII expression in CLL cells where enhanced association of this transcription factor with the PRKCB promoter is likely because of the presence of histone marks permissive of gene activation. We also show how vascular endothelial growth factor (VEGF) regulates PRKCB promoter function in CLL cells, stimulating PKCß gene transcription via increased association of SP1 and decreased association of STAT3. Taken together, these results are the first to demonstrate a clear role for SP1 in the up regulation of PKCßII expression in CLL cells, and the first to link SP1 with the pathogenesis of this and potentially other B cell malignancies where PKCßII is overexpressed.

Targeting B-cell receptor signaling in leukemia and lymphoma: how and why?

B-lymphocytes are dependent on B-cell receptor (BCR) signaling for the constant maintenance of their physiological function, and in many B-cell malignancies this signaling pathway is prone to aberrant activation. This understanding has led to an ever-increasing interest in the signaling networks activated following ligation of the BCR in both normal and malignant cells, and has been critical in establishing an array of small molecule inhibitors targeting BCR-induced signaling. By dissecting how different malignancies signal through BCR, researchers are contributing to the design of more customized therapeutics which have greater efficacy and lower toxicity than previous therapies. This allows clinicians access to an array of approaches to best treat patients whose malignancies have BCR signaling as a driver of pathogenesis.

UHRF1 regulation of the Keap1-Nrf2 pathway in pancreatic cancer contributes to oncogenesis.

The cellular defence protein Nrf2 is a mediator of oncogenesis in pancreatic ductal adenocarcinoma (PDAC) and other cancers. However, the control of Nrf2 expression and activity in cancer is not fully understood. We previously reported the absence of Keap1, a pivotal regulator of Nrf2, in ∼70% of PDAC cases. Here we describe a novel mechanism whereby the epigenetic regulator UHRF1 suppresses Keap1 protein levels. UHRF1 expression was observed in 20% (5 of 25) of benign pancreatic ducts compared to 86% (114 of 132) of pancreatic tumours, and an inverse relationship between UHRF1 and Keap1 levels in PDAC tumours (n = 124) was apparent (p = 0.002). We also provide evidence that UHRF1-mediated regulation of the Nrf2 pathway contributes to the aggressive behaviour of PDAC. Depletion of UHRF1 from PDAC cells decreased growth and enhanced apoptosis and cell cycle arrest. UHRF1 depletion also led to reduced levels of Nrf2-regulated downstream proteins and was accompanied by heightened oxidative stress, in the form of lower glutathione levels and increased reactive oxygen species. Concomitant depletion of Keap1 and UHRF1 restored Nrf2 levels and reversed cell cycle arrest and the increase in reactive oxygen species. Mechanistically, depletion of UHRF1 reduced global and tumour suppressor promoter methylation in pancreatic cancer cell lines, and KEAP1 gene promoter methylation was reduced in one of three cell lines examined. Thus, methylation of the KEAP1 gene promoter may contribute to the suppression of Keap1 protein levels by UHRF1, although our data suggest that additional mechanisms need to be explored. Finally, we demonstrate that K-Ras drives UHRF1 expression, establishing a novel link between this oncogene and Nrf2-mediated cellular protection. Since UHRF1 over-expression occurs in other cancers, its ability to regulate the Keap1-Nrf2 pathway may be critically important to the malignant behaviour of these cancers.

CLL Exosomes Modulate the Transcriptome and Behaviour of Recipient Stromal Cells and Are Selectively Enriched in miR-202-3p.

Bi-directional communication with the microenvironment is essential for homing and survival of cancer cells with implications for disease biology and behaviour. In chronic lymphocytic leukemia (CLL), the role of the microenvironment on malignant cell behaviour is well described. However, how CLL cells engage and recruit nurturing cells is poorly characterised. Here we demonstrate that CLL cells secrete exosomes that are nanovesicles originating from the fusion of multivesicular bodies with the plasma membrane, to shuttle proteins, lipids, microRNAs (miR) and mRNAs to recipient cells. We characterise and confirm the size (50-100 nm) and identity of the CLL-derived exosomes by Electron microscopy (EM), Atomic force microscopy (AFM), flow cytometry and western blotting using both exosome- and CLL-specific markers. Incubation of CLL-exosomes, derived either from cell culture supernatants or from patient plasma, with human stromal cells shows that they are readily taken up into endosomes, and induce expression of genes such as c-fos and ATM as well as enhance proliferation of recipient HS-5 cells. Furthermore, we show that CLL exosomes encapsulate abundant small RNAs and are enriched in certain miRs and specifically hsa-miR-202-3p. We suggest that such specific packaging of miR-202-3p into exosomes results in enhanced expression of 'suppressor of fused' (Sufu), a Hedgehog (Hh) signalling intermediate, in the parental CLL cells. Thus, our data show that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology.

Immunohistochemical analysis indicates that the anatomical location of B-cell non-Hodgkin's lymphoma is determined by differentially expressed chemokine receptors, sphingosine-1-phosphate receptors and integrins.

BACKGROUND: The aim of this study was to elucidate the mechanisms responsible for the location of B-cell non-Hodgkin's lymphoma (B-NHL) at different anatomical sites. We speculated that the malignant B cells in these disorders have the potential for trafficking between blood and secondary lymphoid organs (SLO) or extranodal sites and that their preferential accumulation at different locations is governed by the expression of key molecules that regulate the trafficking of normal lymphocytes. METHODS: Biopsy or blood samples from 91 cases of B-NHL affecting SLO (n = 27), ocular adnexae (n = 51) or blood (n = 13) were analysed by immunohistochemistry or flow cytometry for the expression of the following molecules: CCR7, CCL21 and αL (required for the entry of normal lymphocytes into SLO); CXCR4, CXCL12 and α4 (required for entry into extranodal sites); CXCR5, CXCL13 and S1PR2 (required for tissue retention); S1PR1 and S1PR3 (required for egress into the blood). The expression of each of these molecules was then related to anatomical location and histological subtype. RESULTS: The expression of motility/adhesion molecules varied widely between individual patient samples and correlated much more strongly with anatomical location than with histological subtype. SLO lymphomas [comprising 10 follicular lymphoma (FL), 8 diffuse large B-cell lymphoma (DLBCL), 4 mantle-cell lymphoma (MCL) and 5 marginal-zone lymphoma (MZL)] were characterised by pronounced over-expression of S1PR2, suggesting that the malignant cells in these lymphomas are actively retained at the site of clonal expansion. In contrast, the malignant B cells in ocular adnexal lymphomas (10 FL, 9 DLBCL, 4 MCL and 28 MZL) expressed a profile of molecules suggesting a dynamic process of trafficking involving not only tissue retention but also egress via S1PR3 and homing back to extranodal sites via CXCR4/CXCL12 and α4. Finally, leukaemic lymphomas (6 FL, 5 MCL and 2 MZL) were characterised by aberrant expression of the egress receptor S1PR1 and low expression of molecules required for tissue entry/retention. CONCLUSIONS: In summary, our study strongly suggests that anatomical location in B-NHL is governed by the differential expression of specific adhesion/motility molecules. This novel observation has important implications for therapeutic strategies that aim to disrupt protective micro-environmental interactions.

Expression of functional sphingosine-1 phosphate receptor-1 is reduced by B cell receptor signaling and increased by inhibition of PI3 kinase δ but not SYK or BTK in chronic lymphocytic leukemia cells.

BCR signaling pathway inhibitors such as ibrutinib, idelalisib, and fostamatinib (respective inhibitors of Bruton's tyrosine kinase, PI3Kδ, and spleen tyrosine kinase) represent a significant therapeutic advance in B cell malignancies, including chronic lymphocytic leukemia (CLL). These drugs are distinctive in increasing blood lymphocytes while simultaneously shrinking enlarged lymph nodes, suggesting anatomical redistribution of CLL cells from lymph nodes into the blood. However, the mechanisms underlying this phenomenon are incompletely understood. In this study, we showed that the egress receptor, sphingosine-1-phosphate (S1P) receptor 1 (S1PR1), was expressed at low levels in normal germinal centers and CLL lymph nodes in vivo but became upregulated on normal B cells and, to a variable and lesser extent, CLL cells following in vitro incubation in S1P-free medium. Spontaneous recovery of S1PR1 expression on normal B and CLL cells was prevented by BCR cross-linking, whereas treatment of CLL cells with idelalisib increased S1PR1 expression and migration toward S1P, the greatest increase occurring in cases with unmutated IgH V region genes. Intriguingly, ibrutinib and fostamatinib had no effect on S1PR1 expression or function. Conversely, chemokine-induced migration, which requires integrin activation and is essential for the entry of lymphocytes into lymph nodes as well as their retention, was blocked by ibrutinib and fostamatinib, but not idelalisib. In summary, our results suggest that different BCR signaling inhibitors redistribute CLL cells from lymph nodes into the blood through distinct mechanisms: idelalisib actively promotes egress by upregulating S1PR1, whereas fostamatinib and ibrutinib may reduce CLL cell entry and retention by suppressing chemokine-induced integrin activation.

Does B cell receptor signaling in chronic lymphocytic leukaemia cells differ from that in other B cell types?

Chronic lymphocytic leukaemia (CLL) is an incurable malignancy of mature B cells. CLL is important clinically in Western countries because of its commonality and because of the significant morbidity and mortality associated with the progressive form of this incurable disease. The B cell receptor (BCR) expressed on the malignant cells in CLL contributes to disease pathogenesis by providing signals for survival and proliferation, and the signal transduction pathway initiated by engagement of this receptor is now the target of several therapeutic strategies. The purpose of this review is to outline current understanding of the BCR signal cascade in normal B cells and then question whether this understanding applies to CLL cells. In particular, this review studies the phenomenon of anergy in CLL cells, and whether certain adaptations allow the cells to overcome anergy and allow full BCR signaling to take place. Finally, this review analyzes how BCR signals can be therapeutically targeted for the treatment of CLL.