CUL3 Mutations Research Articles

The heterogeneity of signaling pathways and drug responses in intrahepatic cholangiocarcinoma with distinct genetic mutations

Intrahepatic cholangiocarcinoma (ICC) is the second most common malignancy among primary liver cancers, with an increasing overall incidence and poor prognosis. The intertumoral and intratumoral heterogeneity of ICC makes it difficult to find efficient drug therapies. Therefore, it is essential to identify tumor suppressor genes and oncogenes that induce ICC formation and progression. Here, we performed CRISPR/Cas9-mediated genome-wide screening in a liver-specific Smad4/Pten knockout mouse model (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), which normally generates ICC after 6 months, and detected that mutations in Trp53, Fbxw7, Inppl1, Tgfbr2, or Cul3 markedly accelerated ICC formation. To illustrate the potential mechanisms, we conducted transcriptome sequencing and found that multiple receptor tyrosine kinases were activated, which mainly upregulated the PI3K pathway to induce cell proliferation. Remarkably, the Cul3 mutation stimulated cancer progression mainly by altering the immune microenvironment, whereas other mutations promoted the cell cycle. Moreover, Fbxw7, Inppl1, Tgfbr2, and Trp53 also affect inflammatory responses, apelin signaling, mitotic spindles, ribosome biogenesis, and nucleocytoplasmic transport pathways, respectively. We further examined FDA-approved drugs for the treatment of liver cancer and performed high-throughput drug screening of the gene-mutant organoids. Different drug responses and promising drug therapies, including chemotherapy and targeted drugs, have been discovered for ICC.

KEAP1/NFE2L2 Pathway Signature Outperforms KEAP1/NFE2L2 Mutation Status and Reveals Alternative Pathway-Activating Mutations in NSCLC

IntroductionActivation of the antioxidant KEAP1/NFE2L2 (NRF2) pathway leads to increased glutamine dependence and an aggressive phenotype in NSCLC. Because this pathway has been explored as a clinical target, we developed a transcriptomic signature for identifying KEAP1/NFE2L2-activated tumors. MethodsA total of 971 NSCLC samples were used to train an expression signature (K1N2-score) to predict KEAP1/NFE2L2 mutations. There were 348 in-house NSCLCs that were analyzed using a NanoString expression panel for validation. ResultsThe 46-gene K1N2 score robustly predicted KEAP1/NFE2L2 mutations in the validation set irrespective of histology and mutation (area under the curve: 89.5, sensitivity: 90.2%), suggesting that approximately 90% of KEAP1/NFE2L2 mutations are pathway-activating. The K1N2-score outperformed KEAP1/NFE2L2 mutational status when predicting patient survival (score p = 0.047; mutation p = 0.215). In K1N2 score–positive but KEAP1/NFE2L2 wild-type samples, enrichment testing identified SMARCA4/BRG1 and CUL3 mutations as mimics of KEAP1/NFE2L2 mutations. ConclusionsThe K1N2-score identified KEAP1/NFE2L2-activated NSCLC by robustly detecting KEAP1/NFE2L2mut cases and discovering alternative genomic activators. It is a potential means for selecting patients with a constitutively active KEAP1/NFE2L2 pathway.

Dysregulation of the WNK4-SPAK/OxSR1 pathway has a mild effect on NKCC2 activity

Background: The furosemide-sensitive Na + -K+ - 2Cl − cotransporter (NKCC2) reabsorbs 20% of filtered Na+ along the thick ascending limb (TAL), while and thiazide-sensitive NaCl cotransporter (NCC) reabsorbs 5-10% along the distal convoluted tubule (DCT). The WNK4-SPAK/OxSR1 pathway mediates NKCC2 phosphorylation at threonines 96 and 101 (pT96/pT101) and NCC phosphorylation at threonine 53 (pT53). This activates both cotransporters. A recent study reported that the commonly used pT96/pT101 pNKCC2 antibody cross-reacts with pT53 NCC in mice on the C57BL/6 background due to a 5 amino acid deletion, calling into question some previous findings regarding WNK4-SPAK/OxSR1 regulation of NKCC2. Although WNK4 is highly expressed along both TAL and DCT, dysregulation of WNK4-SPAK/OxSR1 pathway specifically causes Familial Hyperkalemic Hypertension (FHHt), which is exquisitely sensitive to thiazide diuretics, and thus a disease of NCC dysregulation. Using a new pNKCC2 antibody specific in C57BL/6 mice, we tested the hypothesis that the WNK4-SPAK/OxSR1 pathway more strongly activates NCC than it does NKCC2. Methods: A new pT96 NKCC2 antibody was validated in C57BL/6, 129Sv, and Slc12a3 −/− mice. The abundances of pT96 NKCC2 and pT53 NCC were evaluated in Wnk4 −/− , Oxsr1 −/− , Spak −/− , Oxsr1 −/− / Spak −/− mice, and several FHHt model, Cul3 +/−/Δ9 , Klhl3 −/− , and Klhl3 R528H/R528H mice. NKCC2 activity was confirmed by furosemide response test with thiazide pre-treatment in Klhl3 −/− mice. Both male and female were used and all genetically modified strains were on the C57BL/6 background. Results: The pT96 NKCC2 antibody detected pNKCC2 in C57BL/6 mice but not 129Sv mice, and did not cross-react with phosphorylated NCC. pT53 NCC was almost absent but pT96 NKCC2 was only slightly lower in Wnk4 −/− mice. pT53 NCC was almost absent with Spak deletion ( Spak −/− and Oxsr1 −/− / Spak −/− mice) but pT96 NKCC2 abundance did not differ from controls. Oxsr1 deletion ( Oxsr1 −/− and Oxsr1 −/− / Spak −/− mice) led to a modest lowering of pT96 NKCC2/total NKCC2 ratio but not pT96 NKCC2 abundance. Interestingly, immunofluorescence revealed that WNK4 expression was increased along not only DCT but also cortical TAL in Klhl3 −/− mice, but pT96 NKCC2 abundance was not changed. pT53 NCC abundance, but not pT96 NKCC2 abundance, was higher in two other FHHt mouse models with higher WNK4 ( Cul3 +/−/Δ9 and Klhl3 R528H/R528H mice). Consistent with no difference in pT96 NKCC2 abundance, urine Na + excretion after furosemide injection following thiazide treatment was similar between Klhl3 −/− and control mice. Conclusions: NCC is strongly phosphorylated by the WNK4-SPAK pathway, whereas NKCC2 is only mildly phosphorylated by the WNK4-OxSR1 pathway, suggesting another kinase can phosphorylate NKCC2. In FHHt models with Cul3 or Klhl3 mutations, NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide-sensitivity of FHHt. Y.M. received a postdoctoral award from the Uehara Foundation; J.A.M. is funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK098141; M.C.B. is funded by CONACyT, Mexico, Grant 101720. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cullin 3 mutant causing familial hyperkalemic hypertension lacks normal activity in the kidney.

Mutations in the ubiquitin ligase scaffold protein cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). We recently reported that in the kidney, aberrant mutant CUL3 (CUL3-Δ9) activity lowers the abundance of CUL3-Δ9 and Kelch-like 3, the CUL3 substrate adaptor for with-no-lysine kinase 4 (WNK4) and that this is mechanistically important. However, whether CUL3-Δ9 exerts additional effects on other targets that may alter renal function is unclear. Here, we sought to determine 1) whether CUL3-Δ9 expression can rescue the phenotype of renal tubule-specific Cul3 knockout mice, and 2) whether CUL3-Δ9 expression affects other CUL3 substrates. Using an inducible renal tubule-specific system, we studied two CUL3-Δ9-expressing mouse models: Cul3 knockout (Cul3-/-/Δ9) and Cul3 heterozygous background (Cul3+/-/Δ9, FHHt model). The effects of CUL3-Δ9 in these mice were compared with Cul3-/- and Cul3+/- mice. Similar to Cul3-/- mice, Cul3-/-/Δ9 mice displayed polyuria with loss of aquaporin 2 and collecting duct injury; proximal tubule injury also occurred. CUL3-Δ9 did not promote degradation of two CUL3 targets that accumulate in the Cul3-/- kidney: high-molecular-weight (HMW) cyclin E and NAD(P)H:quinone oxidoreductase 1 (NQO1) [a surrogate for the CUL3-Kelch-like ECH-associated protein 1 (KEAP1) substrate nuclear factor erythroid-2-related factor 2]. Since CUL3-Δ9 expression cannot rescue the Cul3-/- phenotype, our data suggest that CUL3-Δ9 cannot normally function in ubiquitin ligase complexes. In Cul3+/-/Δ9 mice, KEAP1 abundance did not differ but NQO1 abundance was higher, suggesting adaptor sequestration by CUL3-Δ9 in vivo. Together, our results provide evidence that in the kidney, CUL3-Δ9 completely lacks normal activity and can trap CUL3 substrate adaptors in inactive complexes.NEW & NOTEWORTHY CUL3 mutation (CUL3-Δ9) causes familial hyperkalemic hypertension (FHHt) by reducing adaptor KLHL3, impairing substrate WNK4 degradation. Whether CUL3-Δ9 affects other targets in kidneys remains unclear. We found that CUL3-Δ9 cannot degrade two CUL3 targets, cyclin E and nuclear factor erythroid-2-related factor 2 (NRF2; using a surrogate marker NQO1), or rescue injury or polyuria caused by Cul3 disruption. In an FHHt model, CUL3-Δ9 impaired NRF2 degradation without reduction of its adaptor KEAP1. Our data provide additional insights into CUL3-Δ9 function in the kidney.

Endothelial Cullin3 Mutation Impairs Nitric Oxide-Mediated Vasodilation and Promotes Salt-Induced Hypertension.

Human hypertension caused by in-frame deletion of CULLIN3 exon-9 (Cul3∆9) is driven by renal and vascular mechanisms. We bred conditionally activatable Cul3∆9 transgenic mice with tamoxifen-inducible Tie2-CREERT2 mice to test the importance of endothelial Cul3. The resultant mice (E-Cul3∆9) trended towards elevated nighttime blood pressure (BP) correlated with increased nighttime activity, but displayed no difference in daytime BP or activity. Male and female E-Cul3∆9 mice together exhibited a decline in endothelial-dependent relaxation in carotid artery. Male but not female E-Cul3∆9 mice displayed severe endothelial dysfunction in cerebral basilar artery. There was no impairment in mesenteric artery and no difference in smooth muscle function, suggesting the effects of Cul3∆9 are arterial bed-specific and sex-dependent. Expression of Cul3∆9 in primary mouse aortic endothelial cells decreased endogenous Cul3 protein, phosphorylated (S1177) endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Cul3∆9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor okadaic acid, but not by a PP1 inhibitor tautomycetin. Because NO deficiency contributes to salt-induced hypertension, we tested the salt-sensitivity of E-Cul3∆9 mice. While both male and female E-Cul3∆9 mice developed salt-induced hypertension and renal injury, the pressor effect of salt was greater in female mutants. The increased salt-sensitivity in female E-Cul3∆9 mice was associated with decreased renovascular relaxation and impaired natriuresis in response to a sodium load. Thus, CUL3 mutations in the endothelium may contribute to human hypertension in part through decreased endothelial NO bioavailability, renovascular dysfunction, and increased salt-sensitivity of BP.

Thioredoxin reductase-1 levels are associated with NRF2 pathway activation and tumor recurrence in non-small cell lung cancer

Activating mutations in the KEAP1/NRF2 pathway characterize a subset of non-small cell lung cancer (NSCLC) associated with chemoresistance and poor prognosis. We herein evaluated the relationship between 64 oxidative stress-related genes and overall survival data from 35 lung cancer datasets. Thioredoxin reductase-1 (TXNRD1) stood out as the most significant predictor of poor outcome. In a cohort of NSCLC patients, high TXNRD1 protein levels correlated with shorter disease-free survival and distal metastasis-free survival post-surgery, including a subset of individuals treated with platinum-based adjuvant chemotherapy. Bioinformatics analysis revealed that NSCLC tumors harboring genetic alterations in the NRF2 pathway (KEAP1, NFE2L2 and CUL3 mutations, and NFE2L2 amplification) overexpress TXNRD1, while no association with EGFR, KRAS, TP53 and PIK3CA mutations was found. In addition, nuclear accumulation of NRF2 overlapped with upregulated TXNRD1 protein in NSCLC tumors. Functional cell assays and gene dependency analysis revealed that NRF2, but not TXNRD1, has a pivotal role in KEAP1 mutant cells’ survival. KEAP1 mutants overexpress TXNRD1 and are less susceptible to the cytotoxic effects of the TXNRD1 inhibitor auranofin when compared to wild-type cell lines. Inhibition of NRF2 with siRNA or ML-385, and glutathione depletion with buthionine-sulfoximine, sensitized KEAP1 mutant A549 cells to auranofin. NRF2 knockdown and GSH depletion also augmented cisplatin cytotoxicity in A549 cells, whereas auranofin had no effect. In summary, these findings suggest that TXNRD1 is not a key determinant of malignant phenotypes in KEAP1 mutant cells, although this protein can be a surrogate marker of NRF2 pathway activation, predicting tumor recurrence and possibly other aggressive phenotypes associated with NRF2 hyperactivation in NSCLC.

Pseudohypoaldosteronism type 2 with CUL3 mutation confirmed following retesting 15 years later

Pseudohypoaldosteronism type 2 with CUL3 mutation confirmed following retesting 15 years later

Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.

Loss-of-function Mutations of CUL3, a High Confidence Gene for Psychiatric Disorders, Lead to Aberrant Neurodevelopment In Human Induced Pluripotent Stem Cells

Both rare, high risk, loss-of-function mutations and common, low risk, genetic variants in the CUL3 gene are strongly associated with neuropsychiatric disorders. Network analyses of neuropsychiatric risk genes have shown high CUL3 expression in the prenatal human brain and an enrichment in neural precursor cells (NPCs) and cortical neurons. The role of CUL3 in human neurodevelopment however, is poorly understood. In the present study, we used CRISPR/Cas9 nickase to knockout CUL3 in human induced pluripotent stem cells (iPSCs). iPSCs were subsequently differentiated into cortical glutamatergic neurons using two different protocols and tested for structural/functional alterations. Immunocytochemical analysis and transcriptomic profiling revealed that pluripotency of heterozygous CUL3 knockout (KO) iPSCs remained unchanged compared to isogenic control iPSCs. Following small molecule-mediated differentiation into cortical glutamatergic neurons however, we detected a significant delay in transition from proliferating radial glia cells/NPCs to postmitotic neurons in CUL3 KO cultures. Notably, direct neural conversion of CUL3 KO iPSCs by lentiviral expression of Neurogenin-2 massively attenuated the neurodevelopmental delay. However, both optogenetic and electrical stimulation of induced neurons revealed decreased excitability in Cullin-3 deficient cultures, while basal synaptic transmission remained unchanged. Analysis of target gene expression pointed to alterations in FGF signaling in CUL3 KO NPCs, which is required for NPC proliferation and self-renewal, while RhoA and Notch signaling appeared unaffected. Our data provide first evidence for a major role of Cullin-3 in neuronal differentiation, and for neurodevelopmental deficits underlying neuropsychiatric disorders associated with CUL3 mutations.

Abstract 1137: Deep targeted and whole exome sequencing identifies driver mutations in the airway epithelium of individuals at high risk for lung cancer

Abstract Insights into tumor mutational burden of the airway epithelium may play important role in stratifying individuals at different levels of risk for lung cancer. Comparison of the genetic profiles of normal airway epithelial cells to tumor mutations can yield valuable information regarding shared mutations as well as presence of neoantigens. We hypothesized that cytologically normal bronchial epithelium of patients with lung cancer and those at high risk for lung cancer harbor driver mutations that can be identified by deep targeted sequencing of selected genes implicated in cancer and whole exome sequencing. We performed next generation sequence-based CAPP-Seq (CAncer Personalized Profiling by deep Sequencing) and whole exome sequencing (WES) of DNA from the bronchial epithelial cells from the bronchial brushings of individuals at-risk (low and high) for lung cancer and patients with lung cancer. Bronchial brushings were collected from consented individuals at the time of bronchoscopy at Vanderbilt University Medical Center and the Nashville Veterans Affairs Medical Center. We used Tammemagi risk model to calculate the risk of developing lung cancer. Lung tumor tissues from matched individuals were also sequenced. Using CAPPSeq, a total of 129 mutations were identified in bronchial brushings. Among individuals without cancer, driver mutations included TP53, PIK3CA, NOTCH1, KRAS, MET, CUL3, NF1, KEAP1 and EGFR mutations in 5 out of 13 high risk and 2 out of 7 low risk individuals. Among individuals with cancer, 13 out of 22 brushings harbored driver mutations in TP53, PIK3CA, ARID1A, PTEN, NOTCH1, KRAS, MET, NFE2L2, CUL3, NF1, and KEAP1 of which mutations in TP53 and PTEN were identified in both tumor and bronchial brushings. At least one mutation was called in 18 brushings of individuals with lung cancer. Mutations detected in both individuals with and without cancer were TP53, PIK3CA, NOTCH1, KRAS, MET, CUL3, and NF1. Using WES on the DNA from the same bronchial brushings (low risk-9; high risk-8 and lung cancer-17), we identified 90 mutations that were also identified by CAPPSeq. Most of the mutations identified by CAPPSeq with >5% variant allelic frequencies (VAF) were also identified by WES. However, WES was not sensitive enough to identify mutations with less than 0.5% VAF. Out of 9 mutations in TP53 identified in the brushings by CAPPSeq, 4 were identified by WES. Mutation in KMTD2, a histone methylation enzyme and believed to be a tumor suppressor, was identified by WES in the brushings and the tumor tissues of patients with lung cancer as well as in one high risk brushings. We identified mutations in cancer driver genes in the bronchial brushings of individuals with and without lung cancer. This field of cancerization effect may provide a tool for risk assessment for lung cancer. This research was funded by U01CA196405 to PPM. Citation Format: SM Jamshedur Rahman, Shih-kai Chu, Shilin Zhao, Yong Zou, Angela Hui, Thomas Stricker, Maximilian Diehn, Pierre P. Massion. Deep targeted and whole exome sequencing identifies driver mutations in the airway epithelium of individuals at high risk for lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1137.

Mouse Models Expressing Mutant CUL3 Provide Insight into the Mechanism of Familial Hyperkalemic Hypertension (FHHT) and Reveal Additional Functional Defects

The pathogenesis of essential hypertension is complex and multifactorial. Studying Mendelian forms of hypertension like Familial Hyperkalemic Hypertension (FHHt) is a powerful approach to identify and understand pathways that may contribute. Cullin‐3 (Cul3) mutations cause the most severe form of FHHt. Cullin 3 (CUL3) along with a substrate adaptor and RING ligase forms a complex that ubiquitinates proteins for proteasomal degradation. In FHHt, mutant CUL3 (CUL3‐D9) activity impairs With‐No‐lysine [K] kinase 4 (WNK4) degradation, leading to increased phosphorylation and thus activation of the Na+ Cl− cotransporter (NCC) along the renal distal convoluted tubule (DCT). We previously reported that CUL3‐D9 degrades the WNK4 adaptor KLHL3 in vitro, and a recent study suggests this occurs in vivo. This depletion of KLHL3 could prevent WNK4 degradation, providing a mechanism by which CUL3‐D9 leads to FHHt. To confirm that CUL3‐D9 degrades KLHL3 in vivo and determine whether it has any other activities, we studied inducible renal epithelial‐specific Cul3 knock‐out (CUL3‐KO) mice and CUL3‐KO mice expressing CUL3‐D9 (CUL3‐KO/D9). Western blotting and immunofluorescence showed that CUL3‐KO mice had higher KLHL3 abundance compared with controls, while CUL3‐KO/D9 had similar KLHL3 abundance to controls. CUL3‐KO and CUL3‐KO/D9 mice also had higher WNK4 abundance compared with controls. These data suggest that KLHL3 is a substrate of wild‐type CUL3, and that CUL3‐D9 is an active form of CUL3 since it can prevent KLHL3 accumulation in CUL3‐KO mice. However, CUL3‐D9 cannot promote WNK4 degradation and did not rescue the chronic kidney disease CUL3‐KO phenotype, suggesting a generalized loss‐of‐function of CUL3‐D9‐containing ubiquitin ligase complexes. A model of FHHt, inducible renal epithelia‐specific heterozygous Cul3 mice with CUL3‐D9 expression (CUL3‐Het/D9), further confirmed this observation with significantly lower KLHL3 abundance compared with control and heterozygous Cul3 mice. To investigate effects of CUL3‐D9 on other CUL3 targets, we investigated the abundance of another CUL3 adaptor protein, Kelch‐like ECH Associated Protein 1 (Keap1), the adaptor for Nuclear factor erythroid 2‐related factor 2 (NRF2). Both CUL3‐KO/D9 and CUL3‐Het/D9 had similar Keap 1 abundance compared with controls, but NAD(P)H Quinone Dehydrogenase 1 (NQO1), a surrogate for NRF2 activity, was significantly higher. These data suggest that in addition to effects on KLHL3/WNK4, CUL3‐D9 forms a dysfunctional complex with Keap1, leading to increased NRF2 activity. In conclusion, we confirm that CUL3‐D9 degrades its adaptor KLHL3 in vivo resulting in WNK4 accumulation, and also show that CUL3‐D9 cannot form normally‐functioning complexes with other substrate adaptors.Support or Funding InformationNIH grants DK098141 and DK117903 to JAM, and AHA 17POST33670206 to MZF

<i>Cul3</i> Regulates Cytoskeleton Protein Homeostasis and Cell Migration During a Critical Window of Brain Development

De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). Here, we used Cul3 mouse models to evaluate the consequences of Cul3 mutations in vivo. Our results show that Cul3 haploinsufficient mice exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. Cul3 mutant brains display cortical lamination abnormalities due to defective neuronal migration and reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal columnar organization, Cul3 haploinsufficiency is associated with decreased spontaneous excitatory and inhibitory activity in the cortex. At the molecular level we show that Cul3 regulates cytoskeletal and adhesion protein abundance in mouse embryos. Abnormal regulation of cytoskeletal proteins in Cul3 mutant neural cells results in atypical organization of the actin mesh at the cell leading edge. Of note, induction of Cul3 haploinsufficiency in adult mice does not result in the behavioral defects observed in constitutive Cul3 haploinsufficient animals. Taken together, our data indicate that Cul3 has a critical role in the regulation of cytoskeletal proteins and neuronal migration and that ASD-associated defects and behavioral abnormalities are primarily due to Cul3 functions at early developmental stages.

Role of KEAP1/NFE2L2 Mutations in the Chemotherapeutic Response of Patients with Non-Small Cell Lung Cancer.

Activation of NFE2L2 has been linked to chemoresistance in cell line models. Recently, somatic mutations that activate NFE2L2, including mutations in NFE2L2, KEAP1, or CUL3, have been found to be associated with poor outcomes in patients with non-small cell lung cancer (NSCLC). However, the impact of these mutations on chemoresistance remains incompletely explored. We investigated the effect of Keap1 deletion on chemoresistance in cell lines from Trp53-based mouse models of lung squamous cell carcinoma (LSCC) and lung adenocarcinoma (LUAD). Separately, we identified 51 patients with stage IV NSCLC with KEAP1, NFE2L2, or CUL3 mutations and a matched cohort of 52 wild-type patients. Time to treatment failure after first-line platinum doublet chemotherapy and overall survival was compared between the two groups. Deletion of Keap1 in Trp53-null murine LUAD and LSCC resulted in increased clonogenic survival upon treatment with diverse cytotoxic chemotherapies. In patients with NSCLC, median time to treatment failure (TTF) after first-line chemotherapy for the KEAP1/NFE2L2/CUL3-mutant cohort was 2.8 months compared with 8.3 months in the control group (P < 0.0001). Median overall survival (OS) was 11.2 months in the KEAP1/NFE2L2/CUL3-mutant group and 36.8 months in the control group (P = 0.006). Keap1 deletion confers chemoresistance in murine lung cancer cells. Patients with metastatic NSCLC with mutations in KEAP1, NFE2L2, or CUL3 have shorter TTF and OS after first-line platinum doublet chemotherapy compared with matched controls. Novel approaches for improving outcomes in this subset of patients with NSCLC are therefore needed.

PIDD interaction with KEAP1 as a new mutation-independent mechanism to promote NRF2 stabilization and chemoresistance in NSCLC

Chemotherapy resistance is a major problem in non-small cell lung cancer (NSCLC) treatment. A major mechanism of chemoresistance involves stabilization of the NRF2 transcription factor. NRF2 levels are normally tightly regulated through interaction with KEAP1, an adaptor that targets NRF2 to the CUL3 E3 ubiquitin ligase for proteolysis. In NSCLC, aberrant NRF2 stabilization is best understood through mutations in NRF2, KEAP1, or CUL3 that disrupt their interaction. Biochemical studies, however, have revealed that NRF2 can also be stabilized through expression of KEAP1-interacting proteins that competitively sequester KEAP1 away from NRF2. Here, we have identified PIDD, as a novel KEAP1-interactor in NSCLC that regulates NRF2. We show that this interaction allows PIDD to reduce NRF2 ubiquitination and increase its stability. We also demonstrate that PIDD promotes chemoresistance in NSCLC cells both in vitro and in vivo, and that this effect is dependent on NRF2. Finally, we report that NRF2 protein expression in a NSCLC cohort exceeds the typical incidence of combined NRF2, KEAP1, and CUL3 mutations, and that NRF2 expression in this cohort is correlated with PIDD levels. Our data identify PIDD as a new NRF2 regulator, and suggest that variations in PIDD levels contribute to differential chemosensitivities among NSCLC patients.

Molecular subtype classification of papillary renal cell cancer using miRNA expression.

BackgroundRenal papillary cell carcinoma (KIRP) is a relatively rare renal malignancy. Although KIRP subtyping about clinical relevance has been defined, there have been scarce number of studies on the molecular characteristics of KIRP subtypes.MethodIn this study, a independent samples t-test was used to identify differentially expressed (DE) miRNAs between tumor and normal samples of KIRP. Meanwhile, we performed unsupervised clustering using DE miRNA expression data to analyze molecular characteristics of KIRP. The Partitioning Around Medoids clustering approach was used to identify molecular subtypes. The cumulative distribution function (CDF), proportion of ambiguously clustered pairs (PAC), principal component analysis (PCA) and consensus heatmaps were used to assess the optimal subtypes. In the differential molecular subtypes, we performed an integrated analysis of survival, DE genes, biological function and somatic mutations on the cohort of KIRP patients from The Cancer Genome Atlas.ResultsFrom solutions with 2, 3, 4, 5, 6 and 7 clusters we selected three KIRP molecular subtypes after assessing PCA, PAC, CDF and consensus heatmaps. We found that the three subtypes are associated with different overall survival and molecular characteristics. Compared with subtype1 and subtype3, subtype2 had a better prognosis in KIRP patients. After exploring their signaling pathways and biological characteristics, we identified the significantly enriched KEGG pathways and Gene Ontology terms for the three subtypes. The distribution of PARD6B, SETD2, STAG2, CUL3, TNRC18, LRBA, IGSF9B and DUNC1H1 mutations differed between the subtypes.ConclusionWe performed unsupervised clustering using differentially expressed miRNA expression data and described the three KIRP molecular subtypes. The three subtypes differed in overall survival, molecular characteristics and gene mutation frequency.

Mechanisms and controversies in mutant Cul3-mediated familial hyperkalemic hypertension.

Autosomal dominant mutations in cullin-3 ( Cul3) cause the most severe form of familial hyperkalemic hypertension (FHHt). Cul3 mutations cause skipping of exon 9, which results in an internal deletion of 57 amino acids from the CUL3 protein (CUL3-∆9). The precise mechanism by which this altered form of CUL3 causes FHHt is controversial. CUL3 is a member of the cullin-RING ubiquitin ligase family that mediates ubiquitination and thus degradation of cellular proteins, including with-no-lysine [K] kinases (WNKs). In CUL3-∆9-mediated FHHt, proteasomal degradation of WNKs is abrogated, leading to overactivation of the WNK targets sterile 20/SPS-1 related proline/alanine-rich kinase and oxidative stress-response kinase-1, which directly phosphorylate and activate the thiazide-sensitive Na+-Cl- cotransporter. Several groups have suggested different mechanisms by which CUL3-∆9 causes FHHt. The majority of these are derived from in vitro data, but recently the Kurz group (Schumacher FR, Siew K, Zhang J, Johnson C, Wood N, Cleary SE, Al Maskari RS, Ferryman JT, Hardege I, Figg NL, Enchev R, Knebel A, O'Shaughnessy KM, Kurz T. EMBO Mol Med 7: 1285-1306, 2015) described the first mouse model of CUL3-∆9-mediated FHHt. Analysis of this model suggested that CUL3-∆9 is degraded in vivo, and thus Cul3 mutations cause FHHt by inducing haploinsufficiency. We recently directly tested this model but found that other dominant effects of CUL3-∆9 must contribute to the development of FHHt. In this review, we focus on our current knowledge of CUL3-∆9 action gained from in vitro and in vivo models that may help unravel this complex problem.

Abstract 3566: The long tail of significantly mutated genes in prostate cancer

Abstract Background: The mutational landscapes of primary and metastatic prostate cancer (PCa) have been robustly analyzed in multiple whole exome sequencing (WES) studies. We hypothesized that an aggregate, uniform analysis of all data generated to date would enable discovery of new significantly mutated genes and pathways not previously associated with PCa, and shed more light onto the genetic differences between primary and metastatic PCa. Methods: We uniformly analyzed WES data of 1,021 tumor and matched germline primary and metastatic PCa (686 primary, 335 metastatic). We performed mutational significance analysis using statistical and biological approaches to determine recurrently altered genes and pathways. Results: We identified 117 significantly mutated genes (Mutsig q&amp;lt;0.1) in PCa. These include epigenetic modifiers [KMT2C (6%), KMT2D (6%), and KDM6A (2.7%)] and regulators of the SWI/SNF complex [SMARCA1 (1.1%), ARID1A (1.5%), ARID1B (1.3%), ARID2 (1.3%), and PBRM1 (0.7%)]. Interestingly, genomic alterations in chromatin remodelers (MLL, SWI/SNF) appear to be significantly mutually exclusive with ETS fusions and SPOP mutations (p&amp;lt;0.001, Fisher’s test), indicating that these mutations may represent a novel distinct oncogenic driver in prostate cancer. Among the novel PCa related pathways, the splicing pathway was found to have oncogenic mutations in key drivers such as SF3B1 (1.1%), U2AF1 (0.5%), and FUBP1 (0.4%), a novel splicing regulator involved in the regulation of MDM2 splicing. We also found truncating mutations in SPEN, a hormone inducible transcriptional repressor, in 2.8% of samples, similar to the frequency observed in breast tumors, and these mutations appear to be significantly associated with AR mutations (p=0.01, Fisher’s test). Our analysis also uncovered mutations in CUL3 (1.3%) and KLHL20, components of an E3-ubiquitin ligase complex that interacts with SPOP to promote the degradation of critical PCa genes (AR, SRC). KLHL20, CUL3, and SPOP mutations are mutually exclusive. Finally, a comparison of primary and metastatic samples identified alterations that are associated with metastatic disease, including AR amplifications and mutations, and loss of TP53, PTEN, and RB1. At lower frequency, metastatic tumors showed enrichment in mutations in MLLs (KMT2C/D), APC, CDK12, BRCA2, CTNNB1, and amplifications of MYC and CCND1. Conclusions: Through aggregation and uniform genomic analysis, we refined the map of somatic mutations in PCa and identified cancer genes and pathways not previously associated with this disease. Our findings may inform patient stratification and translational investigation. Citation Format: Joshua Armenia, Stephanie Mullane, Jianjiong Gao, Debyani Chakravarty, Ritika Kundra, Franklin Huang, Celine Han, Dan Robinson, Levi A. Garraway,, Peter Nelson, Mark Rubin, Mary-Ellen Taplin, Wassim Abida, Charles L. Sawyers, Arul M. Chinnaiyan, Philip W. Kantoff, Johann S. Bono, Nikolaus Schultz, Eliezer M. Van Allen. The long tail of significantly mutated genes in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3566. doi:10.1158/1538-7445.AM2017-3566

Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene

ABSTRACTPseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four different genes: with-no-lysine kinases (WNK) 1 and 4, Kelch-like family member 3 (KLHL3), and cullin 3 (Cul3). Cul3 and KLHL3 form an E3 ligase complex that ubiquitinates and reduces the expression level of WNK4. PHAII-causing mutations in WNK4 and KLHL3 impair WNK4 ubiquitination. However, the molecular pathogenesis of PHAII caused by Cul3 mutations is unclear. In cultured cells and human leukocytes, PHAII-causing Cul3 mutations result in the skipping of exon 9, producing mutant Cul3 protein lacking 57 amino acids. However, whether this phenomenon occurs in the kidneys and is responsible for the pathogenesis of PHAII in vivo is unknown. We generated knock-in mice carrying a mutation in the C-terminus of intron 8 of Cul3, c.1207−1G>A, which corresponds to a PHAII-causing mutation in the human Cul3 gene. Heterozygous Cul3G(−1)A/+ knock-in mice did not exhibit PHAII phenotypes, and the skipping of exon 9 was not evident in their kidneys. However, the level of Cul3 mRNA expression in the kidneys of heterozygous knock-in mice was approximately half that of wild-type mice. Furthermore, homozygous knock-in mice were nonviable. It suggested that the mutant allele behaved like a knockout allele and did not produce Cul3 mRNA lacking exon 9. A reduction in Cul3 expression alone was not sufficient to develop PHAII in the knock-in mice. Our findings highlighted the pathogenic role of mutant Cul3 protein and provided insight to explain why PHAII-causing mutations in Cul3 cause kidney-predominant PHAII phenotypes.

Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia.

Deletion of exon 9 from Cullin-3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin-RING-ubiquitin-ligase complexes. Bound to KLHL3, CUL3-RBX1 ubiquitylates WNK kinases, promoting their ubiquitin-mediated proteasomal degradation. Since WNK kinases activate Na/Cl co-transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin-RING-ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto-ubiquitylates and loses interaction with two important Cullin regulators: the COP9-signalosome and CAND1. A novel knock-in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases.

Hypertension Causing Mutations in Cullin3 Decrease RhoA Ubiquitination and Sequester Substrate Adaptors

Cullin-Ring ubiquitin Ligases (CRL) regulate protein turnover by promoting ubiquitination of substrates. Impaired Cullin3 (Cul3) activity in vascular smooth muscle causes increased RhoA/Rho kinase signaling leading to augmented contraction. PHAII patients with de novo Cul3 mutations which cause skipping of exon 9 (Cul3Δ9) have early onset hypertension, but the mechanistic basis remains unclear. We hypothesize that Cul3Δ9 reduces RhoA ubiquitination and degradation. There was no defect in neddylation of Cul3Δ9. However, binding of Cul3Δ9 to Rbx1, which is necessary for delivering ubiquitin to the substrate was impaired. Consistent with this, ubiquitination of RhoA by Cul3Δ9 was significantly impaired compared with Cul3WT. Thus Cul3Δ9 exhibits impaired ubiquitination of Cul3 substrates. Interestingly, substrate recognition proteins Bacurd1, RhoBTB1 and KLHL3 bound more efficiently to Cul3Δ9 than Cul3WT. Moreover, we observed a significant increase in Cul3Δ9:Cul3WT heterodimers compared to Cul3Δ9:Cul3Δ9 homodimers. This is important because dimerization of CRL complexes is required for efficient ubiquitination activity. Thus Cul3Δ9 may act dominantly by sequestering substrate adaptors from the CRL3 complex or by directly interfering with Cul3WT. We conclude that Cul3Δ9 exhibits impaired ubiquitin ligase activity. In smooth muscle, this would increase the pool of activatable RhoA in response to contractile agonists. When combined with our previous data, we suggest that Cul3-mediated regulation of RhoA protein turnover controls both vascular function and arterial blood pressure. Patients carrying Cul3Δ9 mutations may exhibit elevated smooth muscle RhoA/Rho kinase activity significantly contributing to their hypertension.