Reevaluation of the role of LIP-1 as an ERK/MPK-1 dual specificity phosphatase in the C. elegans germline

The fidelity of a signaling pathway depends on its tight regulation in space and time. Extracellular signal-regulated kinase (ERK) controls wide-ranging cellular processes to promote organismal development and tissue homeostasis. ERK activation depends on a reversible dual phosphorylation on the TEY motif in its active site by ERK kinase (MEK) and dephosphorylation by DUSPs (dual specificity phosphatases). LIP-1, a DUSP6/7 homolog, was proposed to function as an ERK (MPK-1) DUSP in the Caenorhabditis elegans germline primarily because of its phenotype, which morphologically mimics that of a RAS/let-60 gain-of-function mutant (i.e., small oocyte phenotype). Our investigations, however, reveal that loss of lip-1 does not lead to an increase in MPK-1 activity in vivo. Instead, we show that loss of lip-1 leads to 1) a decrease in MPK-1 phosphorylation, 2) lower MPK-1 substrate phosphorylation, 3) phenocopy of mpk-1 reduction-of-function (rather than gain-of-function) allele, and 4) a failure to rescue mpk-1–dependent germline or fertility defects. Moreover, using diverse genetic mutants, we show that the small oocyte phenotype does not correlate with increased ectopic MPK-1 activity and that ectopic increase in MPK-1 phosphorylation does not necessarily result in a small oocyte phenotype. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the C. elegans germline. Our results caution against overinterpretation of the mechanistic underpinnings of orthologous phenotypes, since they may be a result of independent mechanisms, and provide a framework for characterizing the distinct molecular targets through which LIP-1 may mediate its several germline functions.

Vitamin C functions as double-edge sword on cancer progression depending on ERK activation or inhibition mediated by its receptor SVCT2

The effect of Vitamin C (Vc) in oncotherapy was controversial for decades. And hyperactivation of extracellular signal-regulated kinase (ERK) drove tumorigenesis. Herein, we demonstrated that Vc activated ERK through sodium-dependent Vc transporter 2 (SVCT2), while high-dose Vc resulted in persistent ERK feedback inhibition following activation. Extracellular Vc binding to SVCT2 initiated ERK activation, then transmembrane transport of Vc induced dimerization of SVCT2. Activated ERK phosphorylated protein tyrosine phosphatase non-receptor type 12 (PTPN12) at Ser434 and inhibited PTPN12 activity, thus enhancing phosphorylation of Janus kinase 2 (JAK2), which phosphorylated growth factor receptor bound protein 2 (GRB2) at Tyr160 to promote GRB2 dimers dissociation and recruitment of GRB2 to SVCT2, leading to further ERK activation. Different cancers have different sensitivities to Vc, the dose effects of Vc on cancer phenotypes depended on that ERK was activated or inhibited. These findings suggest SVCT2 is a Vc receptor mediating the ERK-PTPN12-JAK2-GRB2-ERK positive feedback loop and a potential target for oncotherapy.

Mechanisms underlying melanoma invasion as a consequence of MLK3 loss

Invasive melanoma is an aggressive form of skin cancer with high incidence of mortality. The process of invasion is a crucial primary step in the metastatic cascade, yet the mechanisms involved are still under investigation. Here we document a critical role for MLK3 (MAP3K11) in the regulation of melanoma cell invasion. We report that cellular loss of MLK3 in melanoma cells promotes cell invasion. Knock down of MLK3 expression results in the hyperactivation of ERK, which is linked to the formation of a BRAF/Hsp90/Cdc37 protein complex. ERK hyperactivation leads to enhanced phosphorylation and inactivation of GSK3β and the stabilization of c-Jun and JNK activity. Blocking of ERK and JNK signaling as well as Hsp90 activity downstream of MLK3-silencing significantly reduces melanoma invasion. Furthermore, our studies show that ERK activation in the aforementioned context is coupled to MT1-MMP transcription as well as the TOM1L1-dependent localization of the membrane protease to invadopodia at the invasive front. These studies provide critical insight into the mechanisms that couple MLK3 loss with BRAF hyperactivation and its consequence on melanoma invasion.

MEK inhibition exerts temporal and myeloid cell-specific effects in the pathogenesis of neurofibromatosis type 1 arteriopathy

Mutations in the NF1 tumor suppressor gene are linked to arteriopathy. Nf1 heterozygosity (Nf1+/–) results in robust neointima formation, similar to humans, and myeloid-restricted Nf1+/– recapitulates this phenotype via MEK-ERK activation. Here we define the contribution of myeloid subpopulations to NF1 arteriopathy. Neutrophils from WT and Nf1+/– mice were functionally assessed in the presence of MEK and farnesylation inhibitors in vitro and neutrophil recruitment to lipopolysaccharide was assessed in WT and Nf1+/– mice. Littermate 12–15 week-old male wildtype and Nf1+/– mice were subjected to carotid artery ligation and provided either a neutrophil depleting antibody (1A8), liposomal clodronate to deplete monocytes/macrophages, or PD0325901 and neointima size was assessed 28 days after injury. Bone marrow transplant experiments assessed monocyte/macrophage mobilization during neointima formation. Nf1+/– neutrophils exhibit enhanced proliferation, migration, and adhesion via p21Ras activation of MEK in vitro and in vivo. Neutrophil depletion suppresses circulating Ly6Clow monocytes and enhances neointima size, while monocyte/macrophage depletion and deletion of CCR2 in bone marrow cells abolish neointima formation in Nf1+/– mice. Taken together, these findings suggest that neurofibromin-MEK-ERK activation in circulating neutrophils and monocytes during arterial remodeling is nuanced and points to important cross-talk between these populations in the pathogenesis of NF1 arteriopathy.

Expression of Spred2 in the urothelial tumorigenesis of the urinary bladder

Aberrant activation of the Ras/Raf/ERK (extracellular-signal-regulated kinase)-MAPK (mitogen-activated protein kinase) pathway is involved in the progression of cancer, including urothelial carcinoma; but the negative regulation remains unclear. In the present study, we investigated pathological expression of Spred2 (Sprouty-related EVH1 domain-containing protein 2), a negative regulator of the Ras/Raf/ERK-MAPK pathway, and the relation to ERK activation and Ki67 index in various categories of 275 urothelial tumors obtained from clinical patients. In situ hybridization demonstrated that Spred2 mRNA was highly expressed in high-grade non-invasive papillary urothelial carcinoma (HGPUC), and the expression was decreased in carcinoma in situ (CIS) and infiltrating urothelial carcinoma (IUC). Immunohistochemically, membranous Spred2 expression, important to interact with Ras/Raf, was preferentially found in HGPUC. Interestingly, membranous Spred2 expression was decreased in CIS and IUC relative to HGPUC, while ERK activation and the expression of the cell proliferation marker Ki67 index were increased. HGPUC with membranous Spred2 expression correlated significantly with lower levels of ERK activation and Ki67 index as compared to those with negative Spred2 expression. Thus, our pathological findings suggest that Spred2 counters cancer progression in non-invasive papillary carcinoma possibly through inhibiting the Ras/Raf/ERK-MAPK pathway, but this regulatory mechanism is lost in cancers with high malignancy. Spred2 appears to be a key regulator in the progression of non-invasive bladder carcinoma.

Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation

Statins represent the most prescribed class of drugs for the treatment of hypercholesterolemia. Effects that go beyond lipid-lowering actions have been suggested to contribute to their beneficial pharmacological properties. Whether and how statins act on macrophages has been a matter of debate. In the present study, we aimed at characterizing the impact of statins on macrophage polarization and comparing these to the effects of bempedoic acid, a recently registered drug for the treatment of hypercholesterolemia, which has been suggested to have a similar beneficial profile but fewer side effects. Treatment of primary murine macrophages with two different statins, i.e., simvastatin and cerivastatin, impaired phagocytotic activity and, concurrently, enhanced pro-inflammatory responses upon short-term lipopolysaccharide challenge, as characterized by an induction of tumor necrosis factor (TNF), interleukin (IL) 1β, and IL6. In contrast, no differences were observed under long-term inflammatory (M1) or anti-inflammatory (M2) conditions, and neither inducible NO synthase (iNOS) expression nor nitric oxide production was altered. Statin treatment led to extracellular-signal regulated kinase (ERK) activation, and the pro-inflammatory statin effects were abolished by ERK inhibition. Bempedoic acid only had a negligible impact on macrophage responses when compared with statins. Taken together, our data point toward an immunomodulatory effect of statins on macrophage polarization, which is absent upon bempedoic acid treatment.

(Pro)renin Receptor Is a Novel Independent Prognostic Marker in Invasive Urothelial Carcinoma of the Bladder

(Pro)renin receptor (PRR) is being investigated in several malignancies as it activates pathogenic pathways that contribute to cell proliferation, immunosuppressive microenvironments, and acquisition of aggressive neoplastic phenotypes. Its implication in urothelial cancer (UC) has not been evaluated so far. We retrospectively evaluate the prognostic role of PRR expression in a series of patients with invasive UC treated with radical cystectomy and other clinical and histopathological parameters including p53, markers of immune-checkpoint inhibition, and basal and luminal phenotypes evaluated by tissue microarray. Cox regression analyses using stepwise selection evaluated candidate prognostic factors and disease-specific survival. PRR was expressed in 77.3% of the primary tumors and in 70% of positive lymph nodes. PRR expression correlated with age (p = 0.006) and was associated with lower preoperatively hemoglobin levels. No other statistical association was evidenced with clinical and pathological variables (gender, ASA score, Charlson comorbidity index, grade, pT, pN) or immunohistochemical expressions evaluated (CK20, GA-TA3, CK5/6, CD44, PD-L1, PD-1, B7-H3, VISTA, and p53). PRR expression in primary tumors was associated with worse survival (log-rank, p = 0.008). Cox regression revealed that PRR expression (HR 1.85, 95% CI 1.22–2.8), pT (HR 7.02, 95% CI 2.68–18.39), pN (HR 2.3, 95% CI 1.27–4.19), and p53 expression (HR 1.95, 95% CI 1.1–3.45) were independent prognostic factors in this series. In conclusion, we describe PRR protein and its prognostic role in invasive UC for the first time. Likely mechanisms involved are MAPK/ERK activation, Wnt/β-catenin signaling, and v-ATPAse function.

Dual BCL-2/BCL-XL Inhibitor Pelcitoclax (APG-1252) Overcomes Intrinsic and Acquired Resistance to Venetoclax in Multiple Myeloma Cells

Multiple myeloma (MM) is marked by several genetic abnormalities, including chromosome translocation t(11;14). Overexpression of anti-apoptotic BCL-2 in t(11;14) MM promotes disease progression, prompting clinical use of the BH3 mimetic and BCL-2 inhibitor venetoclax in combination with proteasome inhibitor therapy. Despite high initial response rates and prolonged progression-free survival, patients commonly relapse. To delineate mechanisms contributing to acquired drug resistance we modeled responses to venetoclax in two highly sensitive MM cell lines (KMS27 and KMS-12PE). Colonies generated from a surviving cell were cultured in high-dose venetoclax to generate monoclonal drug-tolerant expanded persister (DTEP) clones. To determine whether venetoclax resistance in DTEP clones is mediated by transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprogramming, we conducted whole-genome sequencing (WGS) and RNA-seq of the clones. WGS analysis did not show significant differences between parental and resistant clones, but transcriptomic analysis showed shared and unique transcriptome signatures in DTEP clones. Gene set enrichment analysis of the common significantly modulated genes in resistant clones revealed that PKA-ERK-CREB and K-Ras pathway genes were significantly upregulated, whereas apoptotic genes were downregulated in resistant clones compared to parental cells. Importantly, ectopically expressed ERK in venetoclax-sensitive cells conferred a resistant phenotype that was rescued using two specific ERK inhibitors in DTEP clones. These data confirm a key role for ERK activation in acquired venetoclax resistance. Resistant clones were further characterized by reduced mitochondrial priming assessed by dynamic BH3 profiling, with altered expression of anti-apoptotic regulators including MCL-1, BCL-xL, and BCL-W and the replaced BCL-2: BIM complex by both MCL-1 and BCL-xL. Because these data suggested a functional substitution between anti-apoptotic BCL-2 family members in cells with acquired resistance to venetoclax, we next evaluated if MCL-1 or BCL-xL are codependent in MM cells that are insensitive or resistant to venetoclax. Simultaneous inhibition of MCL-1 (via S63845) or BCL-xL (via A155463) and BCL-2 (via venetoclax) increased BIM release and enhanced cell death in resistant clones (vs single agents), with combination index values < 0.3 in all doses. Upregulation of BCL-xL or MCL-1 in MM cells also mediated primary venetoclax resistance independent of genetic hallmarks (e.g. t [11;14]-translocated cells). Thus, simultaneous inhibition of MCL-1 or BCL-xL and BCL-2 triggered synergistic cytotoxicity in MM cell lines intrinsically resistant to venetoclax. These data suggest that combined inhibition of BCL-2 and BCL-xL may overcome venetoclax resistance. However, the dependence of BCL-xL in mature platelets had triggered thrombocytopenia for patients under therapy using BCL-xL inhibitor. To further explore the potential clinical application of targeting BCL-xL, we employed novel BCl-2/BCL-xL dual inhibitor, BH3 mimetic pelcitoclax (APG-1252). Using pro-drug strategy for design, pelcitoclax has limited cell permeability during circulation, and was converted to a more potent metabolite APG-1252-M1 in tumors/tissues. APG-1252-M1 was thus used for in vitro cell based assays. We discovered that APG-1252-M1 induced cytotoxicity in MM cell lines intrinsically resistant to venetoclax (regardless of genetic background or BCL-2:BCL-xL ratio) and also significantly reduced MM cell viability in clones with acquired venetoclax resistance, overcoming ERK activation and decreasing BIM sequestration by BCL-xL. In vivo study using pelcitoclax is ongoing and will be presented at the meeting. In conclusion, we report that venetoclax resistance in MM evolves from outgrowth of persister clones displaying activation of the ERK pathway and a shift in mitochondrial dependency towards BCL-xL, which can potentially be effectively targeted via the novel BCL-2/BCL-xL inhibitor pelcitoclax (APG-1252), which is currently in clinical investigation for solid tumors (NCT03080311). Disclosures Deng: Ascentage Pharma Group: Current Employment. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Novartis: Consultancy; Janssen: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharm: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Amgen: Consultancy; Abbvie: Consultancy; Legend: Consultancy; Pfizer: Consultancy.

ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development in mouse embryos

ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology. Among them, ERBB3 is a binding partner of ERBB2; the ERBB2/3 complex mediates downstream signaling for cell proliferation. ERBB3 has seven consensus binding sites to the p85 regulatory subunit of PI3K, which activates the downstream AKT pathway, leading to the proliferation of various cells. This study generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions. We determined that the proliferation of mesenchymal cells in the atrioventricular (AV) cushion in Erbb3 knock-in mutant embryos was temporarily reduced due to the decrease of AKT and ERK1/2 phosphorylation. Overall, our results suggest that AKT/ERK activation by the ERBB3-dependent PI3K signaling is crucial for AV cushion morphogenesis during embryonic heart development.

Redundant roles of EGFR ligands in the ERK activation waves during collective cell migration

Epidermal growth factor receptor (EGFR) plays a pivotal role in collective cell migration by mediating cell-to-cell propagation of extracellular signal-regulated kinase (ERK) activation. Here, we aimed to determine which EGFR ligands mediate the ERK activation waves. We found that epidermal growth factor (EGF)–deficient cells exhibited lower basal ERK activity than the cells deficient in heparin-binding EGF (HBEGF), transforming growth factor alpha (TGFα) or epiregulin (EREG), but all cell lines deficient in a single EGFR ligand retained the ERK activation waves. Surprisingly, ERK activation waves were markedly suppressed, albeit incompletely, only when all four EGFR ligands were knocked out. Re-expression of the EGFR ligands revealed that all but HBEGF could restore the ERK activation waves. Aiming at complete elimination of the ERK activation waves, we further attempted to knockout NRG1, a ligand for ErbB3 and ErbB4, and found that NRG1-deficiency induced growth arrest in the absence of all four EGFR ligand genes. Collectively, these results showed that EGFR ligands exhibit remarkable redundancy in the propagation of ERK activation waves during collective cell migration.