Morgan O'Hayre

The Emerging Mutational Landscape of G-proteins and G-protein Coupled Receptors in Cancer

Aberrant expression and activity of G proteins and G-protein-coupled receptors (GPCRs) are frequently associated with tumorigenesis. Deep sequencing studies show that 4.2% of tumours carry activating mutations in GNAS (encoding Gαs), and that oncogenic activating mutations in genes encoding Gαq family members (GNAQ or GNA11) are present in ∼66% and ∼6% of melanomas arising in the eye and skin, respectively. Furthermore, nearly 20% of human tumours harbour mutations in GPCRs. Many human cancer-associated viruses also express constitutively active viral GPCRs. These studies indicate that G proteins, GPCRs and their linked signalling circuitry represent novel therapeutic targets for cancer prevention and treatment.

Chronic lymphocytic leukemia cells receive RAF-dependent survival signals in response to CXCL12 that are sensitive to inhibition by sorafenib

The chemokine CXCL12, via its receptor CXCR4, promotes increased survival of chronic lymphocytic leukemia (CLL) B cells that express high levels of ζ-chain-associated protein (ZAP-70), a receptor tyrosine kinase associated with aggressive disease. In this study, we investigated the underlying molecular mechanisms governing this effect. Although significant differences in the expression or turnover of CXCR4 were not observed between ZAP-70(+) and ZAP-70(-) cell samples, CXCL12 induced greater intracellular Ca(2+) flux and stronger and more prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase/ERK kinase (MEK) in the ZAP-70(+) CLL cells. The CXCL12-induced phosphorylation of ERK and MEK in ZAP-70(+) CLL cells was blocked by sorafenib, a small molecule inhibitor of RAF. Furthermore, ZAP-70(+) CLL cells were more sensitive than ZAP-70(-) CLL cells to the cytotoxic effects of sorafenib in vitro at concentrations that can readily be achieved in vivo. The data suggest that ZAP-70(+) CLL cells may be more responsive to survival factors, like CXCL12, that are elaborated by the leukemia microenvironment, and this sensitivity could be exploited for the development of new treatments for patients with this disease. Moreover, sorafenib may have clinical activity for patients with CLL, particularly those with ZAP-70(+) CLL.

Elucidating the CXCL12/CXCR4 Signaling Network in Chronic Lymphocytic Leukemia through Phosphoproteomics Analysis

Background Chronic Lymphocytic Leukemia (CLL) pathogenesis has been linked to the prolonged survival and/or apoptotic resistance of leukemic B cells in vivo, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy. Methodology/Principal Findings To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4), is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27), which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (∼25%) of CLL patients cells examined. Conclusions/Significance Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a previously unknown signaling target of chemokine receptors; therefore, these observations increase our understanding of alternative pathways to migration that may be activated or inhibited by chemokines in the context of cancer cell survival.

Sorafenib-Induced Apoptosis of Chronic Lymphocytic Leukemia Cells Is Associated with Downregulation of RAF and Myeloid Cell Leukemia Sequence 1 (Mcl-1)

We have previously shown that sorafenib, a multikinase inhibitor, exhibits cytotoxic effects on chronic lymphocytic leukemia (CLL) cells. Because the cellular microenvironment can protect CLL cells from drug-induced apoptosis, it is important to evaluate the effect of novel drugs in this context. Here we characterized the in vitro cytotoxic effects of sorafenib on CLL cells and the underlying mechanism in the presence of marrow stromal cells (MSCs) and nurselike cells (NLCs). One single dose of 10 µmol/L or the repeated addition of 1 µmol/L sorafenib caused caspase-dependent apoptosis and reduced levels of phosphorylated B-RAF, C-RAF, extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3) and myeloid cell leukemia sequence 1 (Mcl-1) in CLL cells in the presence of the microenvironment. We show that the RAF/mitogen-activated protein kinase kinase (MEK)/ERK pathway can modulate Mcl-1 expression and contribute to CLL cell viability, thereby associating sorafenib cytotoxicity to its impact on RAF and Mcl-1. To evaluate if the other targets of sorafenib can affect CLL cell viability and contribute to sorafenib-mediated cytotoxicity, we tested the sensitivity of CLL cells to several kinase inhibitors specific for these targets. Our data show that RAF and vascular endothelial growth factor receptor (VEGFR) but not KIT, platelet-derived growth factor receptor (PDGFR) and FMS-like tyrosine kinase 3 (FLT3) are critical for CLL cell viability. Taken together, our data suggest that sorafenib exerts its cytotoxic effect likely via inhibition of the VEGFR and RAF/MEK/ERK pathways, both of which can modulate Mcl-1 expression in CLL cells. Furthermore, sorafenib induced apoptosis of CLL cells from fludarabine refractory patients in the presence of NLCs or MSCs. Our results warrant further clinical exploration of sorafenib in CLL.

Emerging concepts and approaches for chemokine- receptor drug discovery

Importance of the field: Chemokine receptors are most noted for their role in cell migration. However, inappropriate utilization or regulation of these receptors is implicated in many inflammatory diseases, cancer and HIV, making them important drug targets. Areas covered in this review: Allostery, oligomerization and ligand bias are presented as they pertain to chemokine receptors and their associated pathologies. Specific examples of each are described from the recent literature and their implications are discussed in terms of drug discovery efforts targeting chemokine receptors. What the reader will gain: Insight into the expanding view of the multitude of pharmacological variables that need to be considered or that may be exploited in chemokine receptor drug discovery. Take home message: Since 2007, two drugs targeting chemokine receptors have been approved by the FDA, Maraviroc for preventing HIV infection and Mozobil™ for hematopoietic stem cell mobilization. While these successes permit optimism for chemokine receptors as drug targets, only recently has the complexity of this system begun to be appreciated. The concepts of allosteric inhibitors, biased ligands and functional selectivity raise the possibility that drugs with precisely-defined properties can be developed. Other complexities such as receptor oligomerization and tissue-specific functional states of receptors also offer opportunities for increased target and response specificity, although it will be more challenging to translate these ideas into approved therapeutics compared to traditional approaches.

Phosphoproteomic analysis of chemokine signaling networks.

Chemokines induce a number of intracellular signaling pathways by activating second messengers (e.g. calcium) and phosphorylation cascades in order to mediate a myriad of functions including cell migration, survival and proliferation. Although there is some degree of overlap in chemokine receptor-mediated pathway activation, different chemokines will often elicit distinct signaling events. Factors such as cell type, receptor expression levels, G protein availability, and disease state will also influence the signaling response from chemokine-induced receptor activation. Improvements in mass spectrometry, enrichment strategies, and database search programs for identifying phosphopeptides have made phosphoproteomics an accessible biological tool for studying chemokine-induced phosphorylation cascades. Although signaling pathways involved in chemokine-mediated migration have been fairly well characterized, less is known regarding other signaling cascades elicited by chemokines (e.g. to induce proliferation) or the potential for distinct pathway activation in a disease state such as cancer. CXCL12(SDF-1)/CXCR4 signaling has been shown to play an important role in the survival of chronic lymphocytic leukemia (CLL) cells, and thus provides a good system for exploring chemokine signaling, particularly in the interest of survival pathway activation. In this chapter, we describe the use of immobilized metal affinity chromatography (IMAC) phosphopeptide enrichment followed by reversed-phase liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis for exploring CXCL12-mediated signaling in human CLL patient cells.

Mechanisms and consequences of the loss of PHLPP1 phosphatase in chronic lymphocytic leukemia (CLL)

The PHLPP (pleckstrin homology domain and leucine-rich repeat protein phosphatase) family of novel Ser/Thr phosphatases serve as important regulators of cell survival and apoptosis.1, 2 These proposed tumor-suppressors lead to inactivation of Akt, degradation of PKC and negatively regulate ERK1/2 activation.2, 3, 4 Furthermore, reductions in PHLPP1 and/or PHLPP2 expression have been detected in several cancers and linked to cancer progression.4, 5, 6, 7

Abstract 2059: Novel roles for GNA13 and RHOA as tumor suppressor genes

G-protein coupled receptors (GPCRs) and G-proteins are critical signal transduction molecules that regulate cell survival, proliferation, motility and differentiation. However, mutations and disruptions in the expression or function of these molecules have been linked to growth, progression and metastasis of various cancers. Recently, major efforts in the area of genome-wide sequencing of tumors have unveiled numerous previously uncharacterized mutations in G-proteins and GPCRs, which could have significant implications to cancer initiation and progression. In particular, mutations in the gene encoding the G-protein G13 (GNA13) and/or its downstream effector, RhoA, have been identified in B cell lymphomas (particularly Burkitt9s lymphoma and Diffuse Large B cell Lymphoma), T cell lymphomas, and Head and Neck Squamous Cell Carcinomas (HNSCCs). Our analyses indicate that these mutations are highly statistically significant over background cancer gene mutation rate, and mutations in GNA13 alone occur in ∼10-15% of patients with Burkitt9s lymphoma and Diffuse Large B cell Lymphoma. Surprisingly, although G13 and RhoA have previously been linked to cellular transformation and metastatic potential of epithelial cancers, our data suggests that the mutations in GNA13 and/or RHOA in these B cell lymphomas and HNSCC are inhibitory in nature. Furthermore, our results indicate a tumor suppressive function for wild-type GNA13 and RHOA in in vivo cancer models. Overall, our data suggest a novel function tumor suppressive function for the G13/RhoA axis in multiple human malignancies. Citation Format: Morgan O9hayre, Asuka Inoue, Katiuchia Sales, Irina Kufareva, Juan Luis Callejas Valera, Fukun Guo, Constantinos Mikelis, Giovanni DiPasquale, Kira Finkel, Junken Aoki, Yi Zheng, Thomas H. Bugge, J. Silvio Gutkind. Novel roles for GNA13 and RHOA as tumor suppressor genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2015-2059