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.

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.

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.

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.

Variable induction of PRDM1 and differentiation in chronic lymphocytic leukemia is associated with anergy.

Despite antigen engagement and intact B-cell-receptor (BCR) signaling, chronic lymphocytic leukemia (CLL) cells fail to undergo terminal differentiation. We hypothesized that such failure may be due to anergy, as CLL cells exhibit variable levels of nonresponsiveness to surface IgM stimulation that is reversible in vitro. Moreover, anergy is associated with reduced differentiation capacity in normal B cells. We investigated responses of CLL cells to two potent differentiation-promoting agents, IL-21 and cytosine guanine dinucleotide-enriched oligo-deoxynucleotides. The induction of PR domain-containing protein 1 (PRDM1; also known as Blimp-1), a critical regulator of plasmacytic differentiation, by these agents was closely correlated but varied between individual cases, despite functionally intact IL-21 receptor- and Toll-like receptor 9-mediated signal transducer and activator of transcription 3, and nuclear factor-κB pathways. PRDM1 induction was inversely correlated with the extent of anergy as measured by the ability to mobilize intracellular Ca(2+) following BCR crosslinking. PRDM1 responsiveness was associated with other markers of differentiation and proliferation but not with differences in apoptosis. The ability to induce PRDM1 did correlate with differential transcriptional and epigenetic regulation of the PRDM1 gene. These studies extend our understanding of CLL pathobiology, demonstrating that reduced differentiation capacity may be a consequence of anergy. Epigenetic drugs may offer possibilities to reactivate PRDM1 expression as part of novel differentiation therapy approaches.

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.

c-Abl regulates Mcl-1 gene expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a malignancy characterized by clonal expansion of mature B cells that are resistant to apoptosis. This resistance to apoptosis partly results from Mcl-1 expression because high levels of this protein in CLL cells correlate with poor disease prognosis and resistance to chemotherapy. Thus, understanding the mechanism(s) regulating Mcl-1 expression in CLL cells may be useful in the development of new therapies for this incurable disease. In the present study, we show a strong relationship between c-Abl and Mcl-1 expression in CLL cells. We show that treatment of CLL cells with Abl-specific siRNA or with imatinib, to inhibit c-Abl activity, results in the down-regulation of Mcl-1 protein and mRNA. A major regulator of Mcl-1 gene expression is STAT3. Our data show that CLL cells expressing high levels of c-Abl also show elevated levels of phospho-STAT3, and that STAT3 phosphorylation in CLL cells is dependent on c-Abl activity. However, STAT3 phosphorylation by c-Abl requires activation of nuclear factor-κB, secretion of autocrine interleukin-6, and active protein kinase C. Taken together, our data demonstrate the mechanism involved in c-Abl regulation of Mcl-1 expression in CLL cells, and suggest that c-Abl inhibition has therapeutic application in the treatment of this disease.

Cancerous inhibitor of PP2A (CIP2A) at diagnosis of chronic myeloid leukemia is a critical determinant of disease progression.

Prospective identification of patients whose chronic myeloid leukemia (CML) will progress to blast crisis is currently not possible. PP2A is a phosphatase and tumor suppressor that regulates cell proliferation, differentiation, and survival. Cancerous inhibitor of PP2A (CIP2A) is a recently described inhibitor of PP2A in breast and gastric cancer. The aim of this study was to investigate whether CIP2A played a role in CML and whether PP2A or its inhibitor proteins CIP2A or SET could predict clinical outcome. At the time of diagnosis of CML, patients who will later progress to blast crisis have significantly higher levels of CIP2A protein (P < .0001) than patients who do not progress, suggesting that PP2A is functionally inactive. We show that the potential mechanism for disease progression is via altered phosphorylation of the oncogene c-Myc. Knockdown of CIP2A results in increased PP2A activity, decreased c-Myc levels, and a decrease in BCR-ABL1 tyrosine kinase activity. We demonstrate that CIP2A levels at diagnosis can consistently predict patients who will progress to blast crisis. The data show that CIP2A is biologically and clinically important in CML and may be a novel therapeutic target.

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.

Vascular endothelial growth factor stimulates protein kinase CbetaII expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a malignant disease of mature B lymphocytes. We have previously shown that a characteristic feature of CLL cells are high levels of expression and activity of protein kinase CbetaII (PKCbetaII), and that this might influence disease progression by modulating signaling in response to B-cell receptor engagement. The aim of the present work was to investigate the factors involved in stimulating PKCbetaII expression in CLL cells. Here we show that the activation of PKCbetaII in CLL cells stimulated with vascular endothelial growth factor (VEGF) can drive expression of the gene for PKCbeta, PRKCB1. We found that this effect of VEGF on PRKCB1 transcription is paralleled by high expression of PKCbetaII protein and therefore probably contributes to the malignant phenotype of CLL cells. Taken together, the data presented in this study demonstrate that VEGF, in addition to its role in providing prosurvival signals, also plays a role in overexpression of PKCbetaII, an enzyme with a specific pathophysiologic role in CLL.

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.

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.

c-Abl expression in chronic lymphocytic leukemia cells: clinical and therapeutic implications.

c-Abl is important for normal B-cell development, but little is known about the function of this nonreceptor tyrosine kinase in chronic lymphocytic leukemia (CLL). Therefore, the aim of the present study was to examine the clinical, therapeutic, and pathogenetic importance of c-Abl in this disease. We show that the malignant cells of CLL predominantly express the type 1b splice variant of c-Abl and that the expression of c-Abl protein is higher in CLL cells than in normal peripheral blood B cells. Moreover, we show that the levels of c-Abl protein expression correlate positively with tumor burden and disease stage, and negatively with IgV(H) mutation. We also show that STI-571, an inhibitor of c-Abl kinase activity, induces apoptosis of CLL cells with high c-Abl expression levels through a mechanism involving inhibition of nuclear factor kappaB. We conclude that overexpression of c-Abl is likely to play a pathogenetic role in CLL and that STI-571 may be of potential use in the treatment of this disease.

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.

B-cell receptor translocation to lipid rafts and associated signaling differ between prognostically important subgroups of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which interaction of the malignant cells with antigen is thought to play a key role. Individual CLL-cell clones markedly differ in their ability to respond to B-cell receptor ligation, but the mechanism underlying the frequent hyporesponsiveness is incompletely understood. Our aim was to further clarify the extent and cause of the B-cell receptor signaling abnormality in CLL and to assign pathophysiologic relevance to the presence or absence of B-cell receptor responsiveness. We show that extracellular signal-regulated kinase-2 phosphorylation, intracellular Ca2+ increases, CD79a phosphorylation, and translocation of the B-cell receptor to lipid rafts in response to ligation with anti-immunoglobulin M (as a surrogate for antigen) are features of CLL cells with relatively unmutated VH genes (<5% deviation from germ line) and a poor prognosis. B-cell receptor stimulation in these cases also promoted cell survival. In clones with mutated VH genes (>5% deviation from germ line), surface immunoglobulin M ligation failed to induce receptor translocation to rafts or to prolong cell survival. This failure of receptor translocation observed in mutated CLL cells was associated with the constitutive exclusion of the B-cell receptor from rafts by a mechanism involving src-dependent interactions between the B-cell receptor and the actin cytoskeleton. We conclude that exposure to antigen promotes the survival of unmutated CLL clones, contributing to the poor prognosis of this group. In contrast, hyporesponsive mutated CLL clones may have developed into a stage where continuous exposure to antigen results in relative tolerance to antigenic stimulation mediated by the exclusion of the B-cell receptor from lipid rafts.