IκB kinase-α coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy.

Activation of the IκB kinase (IKK) complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-κB has remained elusive. Here, analysis of IKK-dependent substrates in CRC cells after UV treatment revealed that phosphorylation of BRD4 by IKK-α is required for its chromatin-binding at target genes upon DNA damage. Moreover, IKK-α induces the NF-κB-dependent transcription of the cytokine LIF, leading to STAT3 activation, association with BRD4 and recruitment to specific target genes. IKK-α abrogation results in defective BRD4 and STAT3 functions and consequently irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK-α activity, BRD4, and the JAK/STAT pathway enhanced the therapeutic potential of 5-fluorouracil combined with irinotecan in CRC cells and is curative in a chemotherapy-resistant xenograft model. Finally, coordinated expression of LIF and IKK-α is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK-α, BRD4, and JAK/STAT signaling with clinical relevance.

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.

Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB.

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

The MAP1B Binding Domain of Nav1.6 Is Required for Stable Expression at the Axon Initial Segment.

Nav1.6 (SCN8A) is a major voltage-gated sodium channel in the mammalian CNS, and is highly concentrated at the axon initial segment (AIS). As previously demonstrated, the microtubule associated protein MAP1B binds the cytoplasmic N terminus of Nav1.6, and this interaction is disrupted by the mutation p.VAVP(77-80)AAAA. We now demonstrate that this mutation results in WT expression levels on the somatic surface but reduced surface expression at the AIS of cultured rat embryonic hippocampal neurons from both sexes. The mutation of the MAP1B binding domain did not impair vesicular trafficking and preferential delivery of Nav1.6 to the AIS; nor was the diffusion of AIS inserted channels altered relative to WT. However, the reduced AIS surface expression of the MAP1B mutant was restored to WT levels by inhibiting endocytosis with Dynasore, indicating that compartment-specific endocytosis was responsible for the lack of AIS accumulation. Interestingly, the lack of AIS targeting resulted in an elevated percentage of persistent current, suggesting that this late current originates predominantly in the soma. No differences in the voltage dependence of activation or inactivation were detected in the MAP1B binding mutant relative to WT channel. We hypothesize that MAP1B binding to the WT Nav1.6 masks an endocytic motif, thus allowing long-term stability on the AIS surface. This work identifies a critical and important new role for MAP1B in the regulation of neuronal excitability and adds to our understanding of AIS maintenance and plasticity, in addition to identifying new target residues for pathogenic mutations of SCN8ASIGNIFICANCE STATEMENT Nav1.6 is a major voltage-gated sodium channel in human brain, where it regulates neuronal activity due to its localization at the axon initial segment (AIS). Nav1.6 mutations cause epilepsy, intellectual disability, and movement disorders. In the present work, we show that loss of interaction with MAP1B within the Nav1.6 N terminus reduces the steady-state abundance of Nav1.6 at the AIS. The effect is due to increased Nav1.6 endocytosis at this neuronal compartment rather than a failure of forward trafficking to the AIS. This work confirms a new biological role of MAP1B in the regulation of sodium channel localization and will contribute to future analysis of patient mutations in the cytoplasmic N terminus of Nav1.6.

The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.

The voltage-dependent potassium (Kv) channel Kv1.3 regulates leukocyte proliferation, activation, and apoptosis, and altered expression of this channel is linked to autoimmune diseases. Thus, the fine-tuning of Kv1.3 function is crucial for the immune system response. The Kv1.3 accessory protein, potassium voltage-gated channel subfamily E (KCNE) subunit 4, acts as a dominant negative regulatory subunit to both enhance inactivation and induce intracellular retention of Kv1.3. Mutations in KCNE4 also cause immune system dysfunction. Although the formation of Kv1.3-KCNE4 complexes has profound consequences for leukocyte physiology, the molecular determinants involved in the Kv1.3-KCNE4 association are unknown. We now show that KCNE4 associates with Kv1.3 via a tetraleucine motif situated within the carboxy-terminal domain of this accessory protein. This motif would function as an interaction platform, in which Kv1.3 and Ca2+/calmodulin compete for the KCNE4 interaction. Finally, we propose a structural model of the Kv1.3-KCNE4 complex. Our experimental data and the in silico structure suggest that the KCNE4 interaction hides a forward-trafficking motif within Kv1.3 in addition to adding a strong endoplasmic reticulum retention signature to the Kv1.3-KCNE4 complex. Thus, the oligomeric composition of the Kv1.3 channelosome fine-tunes the precise balance between anterograde and intracellular retention elements that control the cell surface expression of Kv1.3 and immune system physiology.-Solé, L., Roig, S. R., Sastre, D., Vallejo-Gracia, A., Serrano-Albarrás, A., Ferrer-Montiel, A., Fernández-Ballester, G., Tamkun, M. M., Felipe, A. The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.

The C-terminal domain of Kv1.3 regulates functional interactions with the KCNE4 subunit.

The voltage-dependent K+ channel Kv1.3 (also known as KCNA3), which plays crucial roles in leukocytes, physically interacts with KCNE4. This interaction inhibits the K+ currents because the channel is retained within intracellular compartments. Thus, KCNE subunits are regulators of K+ channels in the immune system. Although the canonical interactions of KCNE subunits with Kv7 channels are under intensive investigation, the molecular determinants governing the important Kv1.3- KCNE4 association in the immune system are unknown. Our results suggest that the tertiary structure of the C-terminal domain of Kv1.3 is necessary and sufficient for such an interaction. However, this element is apparently not involved in modulating Kv1.3 gating. Furthermore, the KCNE4-dependent intracellular retention of the channel, which negatively affects the activity of Kv1.3, is mediated by two independent and additive mechanisms. First, KCNE4 masks the YMVIEE signature at the C-terminus of Kv1.3, which is crucial for the surface targeting of the channel. Second, we identify a potent endoplasmic reticulum retention motif in KCNE4 that further limits cell surface expression. Our results define specific molecular determinants that play crucial roles in the physiological function of Kv1.3 in leukocytes.

Single-Molecule Imaging of Nav1.6 on the Surface of Hippocampal Neurons Reveals Somatic Nanoclusters.

Voltage-gated sodium (Nav) channels are responsible for the depolarizing phase of the action potential in most nerve cells, and Nav channel localization to the axon initial segment is vital to action potential initiation. Nav channels in the soma play a role in the transfer of axonal output information to the rest of the neuron and in synaptic plasticity, although little is known about Nav channel localization and dynamics within this neuronal compartment. This study uses single-particle tracking and photoactivation localization microscopy to analyze cell-surface Nav1.6 within the soma of cultured hippocampal neurons. Mean-square displacement analysis of individual trajectories indicated that half of the somatic Nav1.6 channels localized to stable nanoclusters ∼230 nm in diameter. Strikingly, these domains were stabilized at specific sites on the cell membrane for >30 min, notably via an ankyrin-independent mechanism, indicating that the means by which Nav1.6 nanoclusters are maintained in the soma is biologically different from axonal localization. Nonclustered Nav1.6 channels showed anomalous diffusion, as determined by mean-square-displacement analysis. High-density single-particle tracking of Nav channels labeled with photoactivatable fluorophores in combination with Bayesian inference analysis was employed to characterize the surface nanoclusters. A subpopulation of mobile Nav1.6 was observed to be transiently trapped in the nanoclusters. Somatic Nav1.6 nanoclusters represent a new, to our knowledge, type of Nav channel localization, and are hypothesized to be sites of localized channel regulation.

Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology.

OBJECTIVE: Voltage-dependent K(+) (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. APPROACH AND RESULTS: Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. CONCLUSIONS: Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.

Does laparoscopic sleeve gastrectomy have any influence on gastroesophageal reflux disease? Preliminary results.

INTRODUCTION: There is no question that Roux-en-Y gastric bypass (RYGB) is the best treatment option for obesity combined with GERD. However, the influence of laparoscopic sleeve gastrectomy (LSG) on this disease remains controversial. It has been said that LSG could induce de novo GERD or worsen it. The aim of our study was to evaluate the influence of LSG on GERD. METHODS AND PROCEDURES: Candidates for LSG underwent esophageal manometry (EM) and 24 h pH monitoring before and 1 year after LSG. Symptoms were evaluated using a validated score. Esophageal function test (EFT's) results and symptoms were compared before and after surgery. RESULTS: Between 4/12 and 9/13, 118 patients underwent LSG. EFT's were performed in 92 (78%) of them preoperatively. From the 19 patients 1 year out of surgery, 14 (73%) completed their EFT's postop. There were 13 women, age 42 ± 12 years, BMI 40 ± 6 kg/m(2). At 14 months, % excess weight loss (EWL) was 74. EM: lower esophageal sphincter (LES) length increased from 2.7 to 3.2 cm (p = NS), and LES pressure decreased from 17.1 to 12.4 mmHg (p ≤ 0.05). Preoperatively, LES was normotensive in 13 (93%) patients; postoperatively, LES was normal in 10 (71%) (p = NS). DeMeester score increased from 12.6 to 28.4 (p ≤ 0.05). Postoperatively, 5 (36%) patients had de novo GERD, in 3 (21%) GERD worsened, 1 (7%) remained with GERD and 5 (36%) remained without reflux. No difference was seen between preop. and postop. symptoms score. CONCLUSION: Our preliminary data showed that after LSG LESP significantly decreased, and the DeMeester score significantly increased. Although LSG results appear appealing in terms of weight loss, patients should be warned that they might need proton pump inhibitors after the operation. Surgeons should probably lower their threshold for indicating RYGB in patients with known preoperative GERD.

Abdominal ultrasound features and reference values in healthy guinea pigs (Cavia porcellus).

BACKGROUND: This study aimed to determine the ultrasonographic features and reference values of the abdominal anatomy in guinea pigs. METHODS: A complete abdominal ultrasonographic examination was performed in 20 adults and 20 young guinea pigs. The thickness of the wall of the gallbladder, stomach, duodenum, caecum, colon and urinary bladder (UB) was measured. Also, the adrenal glands (AGs) (width of the cranial and caudal poles, length), kidneys (length, width, height), ovaries (length, width), testes (length, width), uterus (width) and seminal glands (width) and the thickness of the spleen and pancreas were measured. All the measurements were compared between age groups and sexes. RESULTS: The liver, gallbladder, gastrointestinal tract, pancreas, spleen, kidneys, UB, AGs and great vessels were clearly visualised in all the guinea pigs. No significant statistical differences were found between the sexes, but there were statistically significant differences in the size of the kidneys, AGs, pancreas, spleen and reproductive organs between age groups. No significant differences in the wall thickness of the digestive system, gallbladder and UB were observed between groups. LIMITATIONS: The main limitation of this study is the lack of gross anatomical or histological correlation. CONCLUSIONS: The results of this study support the use of ultrasonography as a diagnostic tool in guinea pigs and provide reference values for the abdominal organs of this species.

Loss of the epithelial marker CDX1 predicts poor prognosis in early-stage CRC patients.

BACKGROUND: We have previously shown that non-curative chemotherapy imposes fetal conversion and high metastatic capacity to cancer cells. From the set of genes differentially expressed in Chemotherapy Resistant Cells, we obtained a characteristic fetal intestinal cell signature that is present in a group of untreated tumors and is sufficient to predict patient prognosis. A feature of this fetal signature is the loss of CDX1. METHODS: We have analyzed transcriptomic data in public datasets and performed immunohistochemistry analysis of paraffin embedded tumor samples from two cohorts of colorectal cancer patients. RESULTS: We demonstrated that low levels of CDX1 are sufficient to identify patients with poorest outcome at the early tumor stages II and III. Presence tumor areas that are negative for CDX1 staining in stage I cancers is associated with tumor relapse. CONCLUSIONS: Our results reveal the actual possibility of incorporating CDX1 immunostaining as a valuable biomarker for CRC patients.

Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas.

Genetic alterations help predict the clinical behavior of diffuse gliomas, but some variability remains uncorrelated. Here, we demonstrate that haploinsufficient deletions of chromatin-bound tumor suppressor NFKB inhibitor alpha (NFKBIA) display distinct patterns of occurrence in relation to other genetic markers and are disproportionately present at recurrence. NFKBIA haploinsufficiency is associated with unfavorable patient outcomes, independent of genetic and clinicopathologic predictors. NFKBIA deletions reshape the DNA and histone methylome antipodal to the IDH mutation and induce a transcriptome landscape partly reminiscent of H3K27M mutant pediatric gliomas. In IDH mutant gliomas, NFKBIA deletions are common in tumors with a clinical course similar to that of IDH wild-type tumors. An externally validated nomogram model for estimating individual patient survival in IDH mutant gliomas confirms that NFKBIA deletions predict comparatively brief survival. Thus, NFKBIA haploinsufficiency aligns with distinct epigenome changes, portends a poor prognosis, and should be incorporated into models predicting the disease fate of diffuse gliomas.

Kinetics and thermodynamics of irreversible inhibition of matrix metalloproteinase 2 by a Co(III) Schiff base complex.

Cobalt(III) Schiff base complexes have been used as potent inhibitors of protein function through the coordination to histidine residues essential for activity. The kinetics and thermodynamics of the binding mechanism of Co(acacen)(NH(3))(2)Cl [Co(acacen); where H(2)acacen is bis(acetylacetone)ethylenediimine] enzyme inhibition has been examined through the inactivation of matrix metalloproteinase 2 (MMP-2) protease activity. Co(acacen) is an irreversible inhibitor that exhibits time- and concentration-dependent inactivation of MMP-2. Co(acacen) inhibition of MMP-2 is temperature-dependent, with the inactivation increasing with temperature. Examination of the formation of the transition state for the MMP-2/Co(acacen) complex was determined to have a positive entropy component indicative of greater disorder in the MMP-2/Co(acacen) complex than in the reactants. With further insight into the mechanism of Co(acacen) complexes, Co(III) Schiff base complex protein inactivators can be designed to include features regulating activity and protein specificity. This approach is widely applicable to protein targets that have been identified to have clinical significance, including matrix metalloproteinases. The mechanistic information elucidated here further emphasizes the versatility and utility of Co(III) Schiff base complexes as customizable protein inhibitors.

Targeting of Kv7.5 (KCNQ5)/KCNE channels to surface microdomains of cell membranes.

BACKGROUND: Kv7.5 (KCNQ5) channels conduct M-type potassium currents in the brain, are expressed in skeletal muscle, and contribute to vascular muscle tone. METHODS: We coexpressed Kv7.5 and KCNE1-3 peptides in HEK293 cells and then analyzed their association using electrophysiology and co-immunoprecipitation, assessed localization using confocal microscopy, examined targeting of the oligomeric channels to cholesterol-rich membrane surface microdomains using lipid raft isolation, and evaluated their membrane dynamics using fluorescence recovery after photobleaching (FRAP). RESULTS: Kv7.5 forms oligomeric channels specifically with KCNE1 and KCNE3. The expression of Kv7.5 targeted to cholesterol-rich membrane surface microdomains was very low. Oligomeric Kv7.5/KCNE1 and Kv7.5/KCNE3 channels did not localize to lipid rafts. However, Kv7.5 association impaired KCNE3 expression in lipid raft microdomains. CONCLUSIONS: Our results indicate that Kv7.5 contributes to the spatial regulation of KCNE3. This new scenario could greatly assist in determining the physiological relevance of putative KCNE3 interactions in nerve and muscle.

Comparison of subcutaneous sedation with alfaxalone or alfaxalone-midazolam in pet guinea pigs (Cavia porcellus) of three different age groups.

OBJECTIVE: To compare the cardiorespiratory effects, quality and duration of sedation of 2 subcutaneous sedation protocols for noninvasive procedures in guinea pigs (GPs). ANIMALS: 24 pet GPs (15 females, 9 males) of 3 different age groups: infant (n = 8), juvenile (8), and adult (8). PROCEDURES: The study design was a randomized, crossover, blinded, clinical trial with a washout period of at least 7 days between protocols. Guinea pigs were sedated SC with alfaxalone (5 mg/kg; group A) or alfaxalone (5 mg/kg) and midazolam (0.5 mg/kg; group A + M) to facilitate blood sampling, radiography, or abdominal ultrasonography. Vital parameters, hemoglobin saturation (SpO2), and sedation scores were recorded every 5 minutes. RESULTS: Mean heart rate was lower in group A than group A + M (P = 0.001), and respiratory rate was significantly (P = 0.001) decreased relative to baseline during sedation in both groups. The SpO2 remained above 95% in both sedation groups. Rectal temperature was significantly (P = 0.001) lower during recovery versus baseline. Onset of sedation was shorter and the duration longer in group A + M than in group A. The duration and depth of the sedation was different between age groups (P = 0.001), being longer and deeper in adults. Bruxism, hectic movements, twitching, and some degree of hyperreactivity were observed during 41 of the 48 sedations. CLINICAL RELEVANCE: Subcutaneous administration of alfaxalone provided reliable sedation for nonpainful procedures in GPs. When combined with midazolam, alfaxalone provided longer and deeper sedation that was more significant in adults than in younger patients.

Does Roux-en-Y Gastric Bypass Really Cure Gastroesophageal Reflux Disease? Analysis of Objective Data.

Background: Laparoscopic Roux-en-Y gastric bypass (LRYGB) is considered the treatment of choice for obesity with gastroesophageal reflux disease (GERD). There are few reports showing objective data based on esophageal function tests (EFTs). The aim of our study was to evaluate the influence of LRYGB on GERD. Methods: Candidates for laparoscopic sleeve gastrectomy (LSG) underwent preoperative esophageal manometry (EM) and 24-hour pH monitoring. Based on the negative influence of LSG on GERD, patients with abnormal pH were offered LRYGB. Those patients repeated EFTs, esophagogastroduodenoscopy, and symptom questionnaire 1 year after surgery. Results: Two hundred fifty LSG candidates underwent preoperative EFTs; 38% were redirected to LRYGB due to abnormal pH and 13 (18%) completed EFTs postoperatively. In ten women, age: 40 ± 7 years, body mass index: 41 ± 1 kg/m2. EM: lower esophageal sphincter (LES) length increased from 2.6 to 2.9 cm (P = not statistically significant [NS]), and LES pressure decreased from 15 to 14.2 mmHg (P = NS). Preoperatively, LES was normotensive in 12 (92%) patients and postoperatively in 11 (85%) (P = NS). DeMeester score decreased from 35.7 to 11 (P < .001). Postoperatively, 9 (69%) patients resolved their GERD, 3 (23%) improved, and 1 (8%) remained the same (P < .001). Symptoms decreased significantly after surgery. Two patients (15%) had Grade A esophagitis. One of them was able to resolve it, while the other 1 remained the same. Conclusions: Our preliminary data showed that after LRYGB, LES pressure remained the same and DeMeester score decreased, while 69% of patients resolved their GERD. Therefore, LRYGB seems to be an excellent option for obesity and GERD.

Sézary syndrome patient-derived models allow drug selection for personalized therapy.

Current therapeutic approaches for Sézary syndrome (SS) do not achieve a significant improvement in long-term survival of patients, and they are mainly focused on reducing blood tumor burden to improve quality of life. Eradication of SS is hindered by its genetic and molecular heterogeneity. Determining effective and personalized treatments for SS is urgently needed. The present work compiles the current methods for SS patient-derived xenograft (PDX) generation and management to provide new perspectives on treatment for patients with SS. Mononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of patients with SS (N = 11). A selected panel of 26 compounds that are inhibitors of the main signaling pathways driving SS pathogenesis, including NF-kB, MAPK, histone deacetylase, mammalian target of rapamycin, or JAK/STAT, was used for in vitro drug sensitivity testing. SS cell viability was evaluated by using the CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated one positive hit using SS patient-derived Sézary cells xenotransplanted (PDX) into NOD-SCID-γ mice. In vitro data indicated that primary malignant SS cells all display different sensitivities against specific pathway inhibitors. In vivo validation using SS PDX mostly reproduced the responses to the histone deacetylase inhibitor panobinostat that were observed in vitro. Our investigations revealed the possibility of using high-throughput in vitro testing followed by PDX in vivo validation for selective targeting of SS tumor cells in a patient-specific manner.

p53 wild-type colorectal cancer cells that express a fetal gene signature are associated with metastasis and poor prognosis.

Current therapy against colorectal cancer (CRC) is based on DNA-damaging agents that remain ineffective in a proportion of patients. Whether and how non-curative DNA damage-based treatment affects tumor cell behavior and patient outcome is primarily unstudied. Using CRC patient-derived organoids (PDO)s, we show that sublethal doses of chemotherapy (CT) does not select previously resistant tumor populations but induces a quiescent state specifically to TP53 wildtype (WT) cancer cells, which is linked to the acquisition of a YAP1-dependent fetal phenotype. Cells displaying this phenotype exhibit high tumor-initiating and metastatic activity. Nuclear YAP1 and fetal traits are present in a proportion of tumors at diagnosis and predict poor prognosis in patients carrying TP53 WT CRC tumors. We provide data indicating the higher efficacy of CT together with YAP1 inhibitors for eradication of therapy resistant TP53 WT cancer cells. Together these results identify fetal conversion as a useful biomarker for patient prognosis and therapy prescription.

KCNE4 suppresses Kv1.3 currents by modulating trafficking, surface expression and channel gating.

Voltage-dependent potassium channels (Kv) play a crucial role in the activation and proliferation of leukocytes. Kv channels are either homo- or hetero-oligomers. This composition modulates their surface expression and serves as a mechanism for regulating channel activity. Kv channel interaction with accessory subunits provides mechanisms for channels to respond to stimuli beyond changes in membrane potential. Here, we demonstrate that KCNE4 (potassium voltage-gated channel subfamily E member 4), but not KCNE2, functions as an inhibitory Kv1.3 partner in leukocytes. Kv1.3 trafficking, targeting and activity are altered by the presence of KCNE4. KCNE4 decreases current density, slows activation, accelerates inactivation, increases cumulative inactivation, retains Kv1.3 in the ER and impairs channel targeting to lipid raft microdomains. KCNE4 associates with Kv1.3 in the ER and decreases the number of Kv1.3 channels at the cell surface, which diminishes cell excitability. Kv1.3 and KCNE4 are differentially regulated upon activation or immunosuppression in macrophages. Thus, lipopolysaccharide-induced activation increases Kv1.3 and KCNE4 mRNA, whereas dexamethasone triggers a decrease in Kv1.3 with no changes in KCNE4. The channelosome composition determines the activity and affects surface expression and membrane localization. Therefore, KCNE4 association might play a crucial role in controlling immunological responses. Our results indicate that KCNE ancillary subunits could be new targets for immunomodulation.

KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology.

The voltage-dependent potassium channel Kv1.3 plays essential roles in the immune system, participating in leukocyte activation, proliferation and apoptosis. The regulatory subunit KCNE4 acts as an ancillary peptide of Kv1.3, modulates K+ currents and controls channel abundance at the cell surface. KCNE4-dependent regulation of the oligomeric complex fine-tunes the physiological role of Kv1.3. Thus, KCNE4 is crucial for Ca2+-dependent Kv1.3-related leukocyte functions. To better understand the role of KCNE4 in the regulation of the immune system, we manipulated its expression in various leukocyte cell lines. Jurkat T lymphocytes exhibit low KCNE4 levels, whereas CY15 dendritic cells, a model of professional antigen-presenting cells, robustly express KCNE4. When the cellular KCNE4 abundance was increased in T cells, the interaction between KCNE4 and Kv1.3 affected important T cell physiological features, such as channel rearrangement in the immunological synapse, cell growth, apoptosis and activation, as indicated by decreased IL-2 production. Conversely, ablation of KCNE4 in dendritic cells augmented proliferation. Furthermore, the LPS-dependent activation of CY15 cells, which induced Kv1.3 but not KCNE4, increased the Kv1.3-KCNE4 ratio and increased the expression of free Kv1.3 without KCNE4 interaction. Our results demonstrate that KCNE4 is a pivotal regulator of the Kv1.3 channelosome, which fine-tunes immune system physiology by modulating Kv1.3-associated leukocyte functions.