IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHCII complexes.

The mechanism by which interferon regulatory factor 8 (IRF8) mutation contributes to lymphomagenesis is unknown. We modeled IRF8 variants in B cell lymphomas and found that they affected the expression of regulators of antigen presentation. Expression of IRF8 mutants in murine B cell lymphomas suppressed CD4, but not CD8, activation elicited by antigen presentation and downmodulated CD74 and human leukocyte antigen (HLA) DM, intracellular regulators of antigen peptide processing/loading in the major histocompatibility complex (MHC) II. Concordantly, mutant IRF8 bound less efficiently to the promoters of these genes. Mice harboring IRF8 mutant lymphomas displayed higher tumor burden and remodeling of the tumor microenvironment, typified by depletion of CD4, CD8, and natural killer cells, increase in regulatory T cells and T follicular helper cells. Deconvolution of bulk RNA sequencing data from IRF8-mutant human diffuse large B cell lymphoma (DLBCL) recapitulated part of the immune remodeling detected in mice. We concluded that IRF8 mutations contribute to DLBCL biology by facilitating immune escape.

IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHC CII complexes.

In diffuse large B-cell lymphoma (DLBCL), the transcription factor IRF8 is the target of a series of potentially oncogenic events, including, chromosomal translocation, focal amplification, and super-enhancer perturbations. IRF8 is also frequently mutant in DLBCL, but how these variants contribute to lymphomagenesis is unknown. We modeled IRF8 mutations in DLBCL and found that they did not meaningfully impact cell fitness. Instead, IRF8 mutants, mapping either to the DNA-binding domain (DBD) or c-terminal tail, displayed diminished transcription activity towards CIITA, a direct IRF8 target. In primary DLBCL, IRF8 mutations were mutually exclusive with mutations in genes involved in antigen presentation. Concordantly, expression of IRF8 mutants in murine B cell lymphomas uniformly suppressed CD4, but not CD8, activation elicited by antigen presentation. Unexpectedly, IRF8 mutation did not modify MHC CII expression on the cell surface, rather it downmodulated CD74 and HLA- DM, intracellular regulators of antigen peptide processing/loading in the MHC CII complex. These changes were functionally relevant as, in comparison to IRF8 WT, mice harboring IRF8 mutant lymphomas displayed a significantly higher tumor burden, in association with a substantial remodeling of the tumor microenvironment (TME), typified by depletion of CD4, CD8, Th1 and NK cells, and increase in T-regs and Tfh cells. Importantly, the clinical and immune phenotypes of IRF8-mutant lymphomas were rescued in vivo by ectopic expression of CD74. Deconvolution of bulk RNAseq data from primary human DLBCL recapitulated part of the immune remodeling detected in mice and pointed to depletion of dendritic cells as another feature of IRF8 mutant TME. We concluded that IRF8 mutations contribute to DLBCL biology by facilitating immune escape.

TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.

A RET::GRB2 fusion in pheochromocytoma defies the classic paradigm of RET oncogenic fusions.

The RET kinase receptor is a target of mutations in neural crest tumors, including pheochromocytomas, and of oncogenic fusions in epithelial cancers. We report a RET::GRB2 fusion in a pheochromocytoma in which RET, functioning as the upstream partner, retains its kinase domain but loses critical C-terminal motifs and is fused to GRB2, a physiological RET interacting protein. RET::GRB2 is an oncogenic driver that leads to constitutive, ligand-independent RET signaling; has transforming capability dependent on RET catalytic function; and is sensitive to RET inhibitors. These observations highlight a new driver event in pheochromocytomas potentially amenable for RET-driven therapy.

Cyclic-AMP signalling, MYC and hypoxia-inducible factor 1α intersect to regulate angiogenesis in B-cell lymphoma.

Angiogenesis and MYC expression associate with poor outcome in diffuse large B-cell lymphoma (DLBCL). MYC promotes neo-vasculature development but whether its deregulation in DLBCL contributes to angiogenesis is unclear. Examination of this relationship may uncover novel pathogenic regulatory circuitry as well as anti-angiogenic strategies in DLBCL. Here, we show that MYC expression positively correlates with vascular endothelial growth factor (VEGF) expression and angiogenesis in primary DLBCL biopsies, independently of dual expressor status or cell-of-origin classification. We found that MYC promotes VEGFA expression, a correlation that was validated in large datasets of mature B-cell tumours. Using DLBCL cell lines and patient-derived xenograft models, we identified the second messenger cyclic-AMP (cAMP) as a potent suppressor of MYC expression, VEGFA secretion and angiogenesis in DLBCL in normoxia. In hypoxia, cAMP switched targets and suppressed hypoxia-inducible factor 1α, a master regulator of VEGFA/angiogenesis in low oxygen environments. Lastly, we used the phosphodiesterase 4b (Pde4b) knockout mouse to demonstrate that the cAMP/PDE4 axis exercises additional anti-angiogenesis by directly targeting the lymphoma microenvironment. In conclusion, MYC could play a direct role in DLBCL angiogenesis, and modulation of cAMP levels, which can be achieved with clinical grade PDE4 inhibitors, has cell and non-cell autonomous anti-angiogenic activity in DLBCL.

MYC, mitochondrial metabolism and O-GlcNAcylation converge to modulate the activity and subcellular localization of DNA and RNA demethylases.

Mitochondria can function as signaling organelles, and part of this output leads to epigenetic remodeling. The full extent of this far-reaching interplay remains undefined. Here, we show that MYC transcriptionally activates IDH2 and increases alpha-ketoglutarate (αKG) levels. This regulatory step induces the activity of αKG-dependent DNA hydroxylases and RNA demethylases, thus reducing global DNA and RNA methylation. MYC, in a IDH2-dependent manner, also promotes the nuclear accumulation of TET1-TET2-TET3, FTO and ALKBH5. Notably, this subcellular movement correlated with the ability of MYC, in an IDH2-dependent manner, and, unexpectedly, of αKG to directly induce O-GlcNAcylation. Concordantly, modulation of the activity of OGT and OGA, enzymes that control the cycling of this non-canonical mono-glycosylation, largely recapitulated the effects of the MYC-IDH2-αKG axis on the subcellular movement of DNA and RNA demethylases. Together, we uncovered a hitherto unsuspected crosstalk between MYC, αKG and O-GlcNAcylation which could influence the epigenome and epitranscriptome homeostasis.

Regulation of PD-L1 expression is a novel facet of cyclic-AMP-mediated immunosuppression.

Cyclic-AMP (cAMP) exerts suppressive effects in the innate and adaptive immune system. The PD-1/PD-L1 immune checkpoint downregulates T-cell activity. Here, we examined if these two immunosuppressive nodes intersect. Using normal and malignant lymphocytes from humans, and the phosphodiesterase 4b (Pde4b) knockout mouse, we found that cAMP induces PD-L1 transcription and protein expression. Mechanistically, we discovered that the cAMP effectors PKA and CREB induce the transcription/secretion of IL-10, IL-8, and IL-6, which initiate an autocrine loop that activates the JAK/STAT pathway and ultimately increase PD-L1 expression in the cell surface. This signaling axis is disarmed at two specific nodes in subsets of diffuse large B-cell lymphoma, which may help explain the variable PD-L1 expression in these tumors. In vivo, we found that despite its immunosuppressive attributes, the PDE4 inhibitor roflumilast did not decrease the clinical activity of checkpoint inhibitors, an important clinical observation given the approved use of these agents in multiple diseases. In summary, we discovered that PD-L1 induction is a part of the repertoire of immunosuppressive actions mediated by cAMP, defined a cytokine-mediated autocrine loop that executes this action and, reassuringly, showed that PDE4 inhibition does not antagonize immune checkpoint blockade in an in vivo syngeneic lymphoma model.

Generation and characterization of the Eµ-Irf8 mouse model.

In mature B-cell malignancies, chromosomal translocations often juxtapose an oncogenic locus to the regulatory regions of the immunoglobulin genes. These genomic rearrangements can associate with specific clinical/pathological sub-entities and inform diagnosis and treatment decisions. Recently, we characterized the t(14;16)(q32;q24) in diffuse large B-cell lymphoma (DLBCL), and showed that it targets the transcription factor IRF8, which is also somatically mutated in ~10% of DLBCLs. IRF8 regulates innate and adaptive immune responses mediated by myeloid/monocytic and lymphoid cells. While the role of IRF8 in human myeloid/dendritic-cell disorders is well established, less is known of its contribution to the pathogenesis of mature B-cell malignancies. To address this knowledge gap, we generated the Eµ-Irf8 mouse model, which mimics the IRF8 deregulation associated with t(14;16) of DLBCL. Eµ-Irf8 mice develop normally and display peripheral blood cell parameters within normal range. However, Eµ-Irf8 mice accumulate pre-pro-B-cells and transitional B-cells in the bone marrow and spleen, respectively, suggesting that the physiological role of Irf8 in B-cell development is amplified. Notably, in Eµ-Irf8 mice, the lymphomagenic Irf8 targets Aicda and Bcl6 are overexpressed in mature B-cells. Yet, the incidence of B-cell lymphomas is not increased in the Eµ-Irf8 model, even though their estimated survival probability is significantly lower than that of WT controls. Together, these observations suggest that the penetrance on the Irf8-driven phenotype may be incomplete and that introduction of second genetic hit, a common strategy in mouse models of lymphoma, may be necessary to uncover the pro-lymphoma phenotype of the Eµ-Irf8 mice.

Functional Characterization of TMEM127 Variants Reveals Novel Insights into Its Membrane Topology and Trafficking.

CONTEXT: TMEM127 is a poorly known tumor suppressor gene associated with pheochromocytomas, paragangliomas, and renal carcinomas. Our incomplete understanding of TMEM127 function has limited our ability to predict variant pathogenicity. PURPOSE: To better understand the function of the transmembrane protein TMEM127 we undertook cellular and molecular evaluation of patient-derived germline variants. DESIGN: Subcellular localization and steady-state levels of tumor-associated, transiently expressed TMEM127 variants were compared to the wild-type protein using immunofluorescence and immunoblot analysis, respectively, in cells genetically modified to lack endogenous TMEM127. Membrane topology and endocytic mechanisms were also assessed. RESULTS: We identified 3 subgroups of mutations and determined that 71% of the variants studied are pathogenic or likely pathogenic through loss of membrane-binding ability, stability, and/or internalization capability. Investigation into an N-terminal cluster of missense variants uncovered a previously unrecognized transmembrane domain, indicating that TMEM127 is a 4- transmembrane, not a 3-transmembrane domain-containing protein. Additionally, a C-terminal variant with predominant plasma membrane localization revealed an atypical, extended acidic, dileucine-based motif required for TMEM127 internalization through clathrin-mediated endocytosis. CONCLUSION: We characterized the functional deficits of several germline TMEM127 variants and identified novel structure-function features of TMEM127. These findings will assist in determining pathogenicity of TMEM127 variants and will help guide future studies investigating the cellular role of TMEM127.

MYC Regulation of D2HGDH and L2HGDH Influences the Epigenome and Epitranscriptome.

Mitochondrial D2HGDH and L2HGDH catalyze the oxidation of D-2-HG and L-2-HG, respectively, into αKG. This contributes to cellular homeostasis in part by modulating the activity of αKG-dependent dioxygenases. Signals that control the expression/activity of D2HGDH/L2HGDH are presumed to broadly influence physiology and pathology. Using cell and mouse models, we discovered that MYC directly induces D2HGDH and L2HGDH transcription. Furthermore, in a manner suggestive of D2HGDH, L2HGDH, and αKG dependency, MYC activates TET enzymes and RNA demethylases, and promotes their nuclear localization. Consistent with these observations, in primary B cell lymphomas MYC expression positively correlated with enhancer hypomethylation and overexpression of lymphomagenic genes. Together, these data provide additional evidence for the role of mitochondria metabolism in influencing the epigenome and epitranscriptome, and imply that in specific contexts wild-type TET enzymes could demethylate and activate oncogenic enhancers.

The tumor suppressor TMEM127 regulates insulin sensitivity in a tissue-specific manner.

Understanding the molecular components of insulin signaling is relevant to effectively manage insulin resistance. We investigated the phenotype of the TMEM127 tumor suppressor gene deficiency in vivo. Whole-body Tmem127 knockout mice have decreased adiposity and maintain insulin sensitivity, low hepatic fat deposition and peripheral glucose clearance after a high-fat diet. Liver-specific and adipose-specific Tmem127 deletion partially overlap global Tmem127 loss: liver Tmem127 promotes hepatic gluconeogenesis and inhibits peripheral glucose uptake, while adipose Tmem127 downregulates adipogenesis and hepatic glucose production. mTORC2 is activated in TMEM127-deficient hepatocytes suggesting that it interacts with TMEM127 to control insulin sensitivity. Murine hepatic Tmem127 expression is increased in insulin-resistant states and is reversed by diet or the insulin sensitizer pioglitazone. Importantly, human liver TMEM127 expression correlates with steatohepatitis and insulin resistance. Our results suggest that besides tumor suppression activities, TMEM127 is a nutrient-sensing component of glucose/lipid homeostasis and may be a target in insulin resistance.

Synonymous but Not Silent: A Synonymous VHL Variant in Exon 2 Confers Susceptibility to Familial Pheochromocytoma and von Hippel-Lindau Disease.

CONTEXT: von Hippel-Lindau (VHL) disease, comprising renal cancer, hemangioblastoma, and/or pheochromocytoma (PHEO), is caused by missense or truncating variants of the VHL tumor-suppressor gene, which is involved in degradation of hypoxia-inducible factors (HIFs). However, the role of synonymous VHL variants in the disease is unclear. OBJECTIVE: We evaluated a synonymous VHL variant in patients with familial PHEO or VHL disease without a detectable pathogenic VHL mutation. DESIGN: We performed genetic and transcriptional analyses of leukocytes and/or tumors from affected and unaffected individuals and evaluated VHL splicing in existing cancer databases. RESULTS: We identified a synonymous VHL variant (c.414A>G, p.Pro138Pro) as the driver event in five independent individuals/families with PHEOs or VHL syndrome. This variant promotes exon 2 skipping and hence, abolishes expression of the full-length VHL transcript. Exon 2 spans the HIF-binding domain required for HIF degradation by VHL. Accordingly, PHEOs carrying this variant display HIF hyperactivation typical of VHL loss. Moreover, other exon 2 VHL variants from the The Cancer Genome Atlas pan-cancer datasets are biased toward expression of a VHL transcript that excludes this exon, supporting a broader impact of this spliced variant. CONCLUSION: A recurrent synonymous VHL variant (c.414A>G, p.Pro138Pro) confers susceptibility to PHEO and VHL disease through splice disruption, leading to VHL dysfunction. This finding indicates that certain synonymous VHL variants may be clinically relevant and should be considered in genetic testing and surveillance settings. The observation that other coding VHL variants can exclude exon 2 suggests that dysregulated splicing may be an underappreciated mechanism in VHL-mediated tumorigenesis.

Synergistic Targeting of the Regulatory and Catalytic Subunits of PI3Kδ in Mature B-cell Malignancies.

Purpose: Aberrant activation of the B-cell receptor (BCR) is implicated in the pathogenesis of mature B-cell tumors, a concept validated in part by the clinical success of inhibitors of the BCR-related kinases BTK (Bruton's tyrosine kinase) and PI3Kδ. These inhibitors have limitations, including the paucity of complete responses, acquired resistance, and toxicity. Here, we examined the mechanism by which the cyclic-AMP/PDE4 signaling axis suppresses PI3K, toward identifying a novel mechanism-based combinatorial strategy to attack BCR-dependency in mature B-cell malignancies.Experimental Design: We used in vitro and in vivo diffuse large B-cell lymphoma (DLBCL) cell lines and primary chronic lymphocytic leukemia (CLL) samples to preclinically evaluate the effects of the combination of the FDA-approved phosphodiesterase 4 (PDE4) inhibitor roflumilast and idelalisib on cell survival and tumor growth. Genetic models of gain- and loss-of-function were used to map multiple signaling intermediaries downstream of the BCR.Results: Roflumilast elevates the intracellular levels of cyclic-AMP and synergizes with idelalisib in suppressing tumor growth and PI3K activity. Mechanistically, we show that roflumilast suppresses PI3K by inhibiting BCR-mediated activation of the P85 regulatory subunit, distinguishing itself from idelalisib, an ATP-competitive inhibitor of the catalytic P110 subunit. Using genetic models, we linked the PDE4-regulated modulation of P85 activation to the oncogenic kinase SYK.Conclusions: These data demonstrate that roflumilast and idelalisib suppress PI3K by distinct mechanisms, explaining the basis for their synergism, and suggest that the repurposing of PDE4 inhibitors to treat BCR-dependent malignancies is warranted. Clin Cancer Res; 24(5); 1103-13. ©2017 AACR.

Biology Informs Treatment Choices in Diffuse Large B Cell Lymphoma.

The effective deployment of rationally developed therapies for diffuse large B cell lymphoma (DLBCL) requires rapid assimilation of new biological data. Within this framework, here we address topical issues at the intersection of DLBCL biology and the clinic. We discuss targeting of B cell receptor (BCR) signaling, with emphasis on identifying patients who may benefit from this maneuver and how to best achieve it. We address strategies to modulate the DLBCL microenvironment, including the use of immune checkpoint inhibitors in selected DLBCL subsets, and the potential activity of alternative antiangiogenic therapies. Lastly, we highlight the emerging recognition of MYC and BCL2 coexpression as the most robust predictor of DLBCL outcome, and discuss rationally conceived experimental approaches to treat these high-risk patients.

Tenovin-6 inhibits proliferation and survival of diffuse large B-cell lymphoma cells by blocking autophagy.

Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive non-Hodgkin lymphomas. It is curable but one-third of cases are refractory to therapy or relapse after initial response highlighting the urgent need for developing novel therapeutic approaches. Targeting sirtuins, particularly SIRT1 by genetic approaches or using pharmaceutical inhibitor tenovin-6, has shown promising therapeutic potential in various hematopoietic malignancies. However, it remains unknown whether these approaches are effective for DLBCL. In this study, we have found that tenovin-6 potently inhibits the proliferation and survival of DLBCL cells. Surprisingly, specific knockdown of SIRT1/2/3 has no effect on DLBCL. Mechanistically, tenovin-6 increases the level of microtubule-associated protein 1 light chain 3B (LC3B)-II in a SIRT1/2/3- and p53-independent manner in DLBCL cell lines. Tenovin-6-mediated increase of LC3B-II is through inhibition of classical autophagy pathway. Furthermore, inhibition of the autophagy pathway by using other inhibitors or by knocking down key genes in the pathway impairs cell proliferation and survival of DLBCL cells. These results indicate that targeting the autophagic pathway could be a novel therapeutic strategy for DLBCL and that precaution should be taken to interpret data where tenovin-6 was used as an inhibitor of sirtuins.

Safety and Pharmacodynamics of the PDE4 Inhibitor Roflumilast in Advanced B-cell Malignancies.

Purpose: In this study, we aimed to validate our extensive preclinical data on phosphodiesterase 4 (PDE4) as actionable target in B-cell malignancies. Our specific objectives were to determine the safety, pharmacokinetics, and pharmacodynamics (PI3K/AKT activity), as well as to capture any potential antitumor activity of the PDE4 inhibitor roflumilast in combination with prednisone in patients with advanced B-cell malignancies.Experimental Design: Single-center, exploratory phase Ib open-label, nonrandomized study. Roflumilast (500 mcg PO) was given daily for 21 days with prednisone on days 8 to 14. Additional 21-day cycles were started if patients tolerated cycle 1 and had at least stable disease.Results: Ten patients, median age 65 years with an average of three prior therapies, were enrolled. The median number of cycles administered was 4 (range, 1-13). Treatment was well tolerated; the most common ≥grade 2 treatment-related adverse events were fatigue, anorexia (≥25%), and transient ≥ grade 2 neutropenia (30%). Treatment with roflumilast as a single agent significantly suppressed PI3K activity in the 77% of patients evaluated; on average, patients with PI3K/AKT suppression stayed in trial for 156 days (49-315) versus 91 days (28-139 days) for those without this biomarker response. Six of the nine evaluable patients (66%) had partial response or stable disease. The median number of days in trial was 105 days (range, 28-315).Conclusions: Repurposing the PDE4 inhibitor roflumilast for treatment of B-cell malignancies is safe, suppresses the oncogenic PI3K/AKT kinases, and may be clinically active. Clin Cancer Res; 23(5); 1186-92. ©2016 AACR.

Phosphodiesterase 4 inhibitors have wide-ranging activity in B-cell malignancies.

Phosphodiesterase 4 (PDE4) inhibition restores the suppressive effects of 3',5'-cyclic adenosine monophosphate in lymphocytes. In this concise review, we detail how PDE4 inhibition downmodulates the B-cell receptor (BCR)-related kinases spleen tyrosine kinase and phosphatidylinositol 3-kinase and inhibits vascular endothelial growth factor A secretion by tumor cells, inducing cancer cell apoptosis and blocking angiogenesis in the microenvironment. We describe the successful clinical repurposing of PDE4 inhibitors in B-cell malignancies, and propose that given their anti-inflammatory/immunomodulatory activity, these agents will suppress BCR signals without the toxicity associated with other targeted biological doublets.

A phosphodiesterase 4B-dependent interplay between tumor cells and the microenvironment regulates angiogenesis in B-cell lymphoma.

Angiogenesis associates with poor outcome in diffuse large B-cell lymphoma (DLBCL), but the contribution of the lymphoma cells to this process remains unclear. Addressing this knowledge gap may uncover unsuspecting proangiogenic signaling nodes and highlight alternative antiangiogenic therapies. Here, we identify the second messenger cyclic-AMP (cAMP) and the enzyme that terminates its activity, phosphodiesterase 4B (PDE4B), as regulators of B-cell lymphoma angiogenesis. We first show that cAMP, in a PDE4B-dependent manner, suppresses PI3K/AKT signals to downmodulate vascular endothelial growth factor (VEGF) secretion and vessel formation in vitro. Next, we create a novel mouse model that combines the lymphomagenic Myc transgene with germline deletion of Pde4b. We show that lymphomas developing in a Pde4b-null background display significantly lower microvessel density (MVD) in association with lower VEGF levels and PI3K/AKT activity. We recapitulate these observations by treating lymphoma-bearing mice with the FDA-approved PDE4 inhibitor, Roflumilast. Lastly, we show that primary human DLBCLs with high PDE4B expression display significantly higher MVD. Here, we defined an unsuspected signaling circuitry in which the cAMP generated in lymphoma cells downmodulates PI3K/AKT and VEGF secretion to negatively influence vessel development in the microenvironment. These data identify PDE4 as an actionable antiangiogenic target in DLBCL.

Recurrent Mutations of Chromatin-Remodeling Genes and Kinase Receptors in Pheochromocytomas and Paragangliomas.

PURPOSE: Pheochromocytomas and paragangliomas (PPGL) are genetically heterogeneous tumors of neural crest origin, but the molecular basis of most PPGLs is unknown. EXPERIMENTAL DESIGN: We performed exome or transcriptome sequencing of 43 samples from 41 patients. A validation set of 136 PPGLs was used for amplicon-specific resequencing. In addition, a subset of these tumors was subjected to microarray-based transcription, protein expression, and histone methylation analysis by Western blotting or immunohistochemistry. In vitro analysis of mutants was performed in cell lines. RESULTS: We detected mutations in chromatin-remodeling genes, including histone-methyltransferases, histone-demethylases, and histones in 11 samples from 8 patients (20%). In particular, we characterized a new cancer syndrome involving PPGLs and giant cell tumors of bone (GCT) caused by a postzygotic G34W mutation of the histone 3.3 gene, H3F3A Furthermore, mutations in kinase genes were detected in samples from 15 patients (37%). Among those, a novel germline kinase domain mutation of MERTK detected in a patient with PPGL and medullary thyroid carcinoma was found to activate signaling downstream of this receptor. Recurrent germline and somatic mutations were also detected in MET, including a familial case and sporadic PPGLs. Importantly, in each of these three genes, mutations were also detected in the validation group. In addition, a somatic oncogenic hotspot FGFR1 mutation was found in a sporadic tumor. CONCLUSIONS: This study implicates chromatin-remodeling and kinase variants as frequent genetic events in PPGLs, many of which have no other known germline driver mutation. MERTK, MET, and H3F3A emerge as novel PPGL susceptibility genes. Clin Cancer Res; 22(9); 2301-10. ©2015 AACR.