Therapeutics such as lapatinib that target ERBB2 often provide initial clinical benefit but resistance frequently develops. Adaptive responses leading to lapatinib resistance involve reprogramming of ...the kinome through reactivation of ERBB2/ERBB3 signaling and transcriptional upregulation and activation of multiple tyrosine kinases. The heterogeneity of induced kinases prevents their targeting by a single kinase inhibitor, underscoring the challenge of predicting effective kinase inhibitor combination therapies. We hypothesized that to make the tumor response to single kinase inhibitors durable, the adaptive kinome response itself must be inhibited. Genetic and chemical inhibition of BET bromodomain chromatin readers suppresses transcription of many lapatinib-induced kinases involved in resistance including ERBB3, IGF1R, DDR1, MET, and FGFRs, preventing downstream SRC/FAK signaling and AKT reactivation. Combining inhibitors of kinases and chromatin readers prevents kinome adaptation by blocking transcription, generating a durable response to lapatinib and overcoming the dilemma of heterogeneity in the adaptive response.
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Lapatinib induces heterogeneous RTK-based kinome adaptation in ERBB2+ cellsMultiple unrelated kinases contribute to cell growth in the presence of lapatinibBET bromodomain inhibition suppresses expression of lapatinib-induced kinasesTargeting kinome adaptation makes kinase inhibition durable
Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition ...in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.
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► Inhibition of the MEK-ERK pathway rapidly reprograms kinome activity in tumors ► c-Myc degradation induces expression and activation of receptor tyrosine kinases ► Receptor tyrosine kinase activation overcomes MEK inhibition, causing resistance ► Kinome inhibitor response profiling rationally predicts combination therapies
Chemical proteomics reveals the dynamic rewiring of kinase networks in response to treatment with MEK inhibitors, uncovering how resistance to these inhibitors emerges in tumors and highlighting combination therapy approaches for bypassing drug resistance.
Epithelial stem cells self-renew while maintaining multipotency, but the dependence of stem cell properties on maintenance of the epithelial phenotype is unclear. We previously showed that ...trophoblast stem (TS) cells lacking the protein kinase MAP3K4 maintain properties of both stemness and epithelial-mesenchymal transition (EMT). Here, we show that MAP3K4 controls the activity of the histone acetyltransferase CBP, and that acetylation of histones H2A and H2B by CBP is required to maintain the epithelial phenotype. Combined loss of MAP3K4/CBP activity represses expression of epithelial genes and causes TS cells to undergo EMT while maintaining their self-renewal and multipotency properties. The expression profile of MAP3K4-deficient TS cells defines an H2B acetylation-regulated gene signature that closely overlaps with that of human breast cancer cells. Taken together, our data define an epigenetic switch that maintains the epithelial phenotype in TS cells and reveals previously unrecognized genes potentially contributing to breast cancer.
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► TS cells lacking MAP3K4 activity (TSKI4) exhibit properties of stemness and EMT ► Loss of H2A/H2B acetylation by CBP induces EMT, but does not affect stemness ► TS cells maintain epithelial identity and stem cell renewal by independent mechanisms ► TSKI4 cells and human breast tumors share an intersecting EMT gene signature
A history of gastrointestinal bleeding (GIB) in patients with atrial fibrillation (AF) may impact decisions about anticoagulation treatment. We sought to determine whether prior GIB in patients with ...AF taking anticoagulants was associated with an increased risk of stroke or major hemorrhage.
We analyzed key efficacy and safety outcomes in patients with prior GIB in ARISTOTLE. Centrally adjudicated outcomes according to GIB history were analyzed using Cox proportional hazards models adjusted for randomized treatment and established risk factors.
A total of 784 (4.3%) patients had prior GIB events (321 41% lower, 463 59% upper); 215 (27%) occurred <1 year before study enrollment. Patients with prior GIB were older, had more comorbidities, and higher CHADS2 and HAS-BLED scores than those with no GIB. Major GIB occurred more frequently in those with prior GIB (lower: aHR 1.72, 95% CI 0.86-3.42; upper: aHR 3.13, 95% CI 1.97-4.96). This association with major GIB was more pronounced in patients with GIB <1 year before randomization versus no recent GIB (recent lower: aHR 2.58, 95% CI 0.95-7.01; recent upper: aHR 5.16, 95% CI 2.66-10.0). There was no association between prior GIB and risk of stroke/systemic embolism or all-cause death. In those with prior GIB, the apixaban versus warfarin relative risks for stroke/systemic embolism, hemorrhagic stroke, death, or major bleeding were consistent with the results of the overall trial.
In patients with AF on oral anticoagulants, prior GIB was associated with an increased risk of subsequent major GIB but not stroke, intracranial bleeding, or all-cause mortality. For the key outcomes of stroke, hemorrhagic stroke, death, and major bleeding, we found no evidence that the treatment effect (apixaban vs. warfarin) was modified by a history of GIB.
The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, ...requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent K m values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 μM. DHA was also strongly preferred by ACSL6_v2. The apparent K m value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.
The MAPK kinase kinase MEKK4 is required for neurulation and skeletal patterning during mouse development. MEKK4 phosphorylates and activates MKK4/MKK7 and MKK3/MKK6 leading to the activation of JNK ...and p38, respectively. MEKK4 is believed to be auto-inhibited, and its interaction with other proteins controls its dimerization and activation. TRAF4, GADD45, and Axin each bind and activate MEKK4, with TRAF4 and Axin binding to the kinase domain and GADD45 binding within the N-terminal regulatory domain. Here we show that similar to the interaction with TRAF4 and Axin, the kinase domain of MEKK4 interacts with the multifunctional serine/threonine kinase GSK3β. GSK3β binding to MEKK4 blocks MEKK4 dimerization that is required for MEKK4 activation, effectively inhibiting MEKK4 stimulation of the JNK and p38 MAPK pathways. Inhibition of GSK3β kinase activity with SB216763 results in enhanced MEKK4 kinase activity and increased JNK and p38 activation, indicating that an active state of GSK3β is required for binding and inhibition of MEKK4 dimerization. Furthermore, GSK3β phosphorylates specific serines and threonines in the N terminus of MEKK4. Together, these findings demonstrate that GSK3β binds to the kinase domain of MEKK4 and regulates MEKK4 dimerization. However, unlike TRAF4, Axin, and GADD45, GSK3β inhibits MEKK4 activity and prevents its activation of JNK and p38. Thus, control of MEKK4 dimerization is regulated both positively and negatively by its interaction with specific proteins.
Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC 6.2.1.3 ) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the ...subcellular
locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting
peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated
membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located
in mitochondrial membrane. Consistent with these locations, N -ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a
second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive
inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting
that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently
by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding
normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5â2.0-fold,
consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken
together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS
isoforms in unique subcellular locations.
MEK1/2 inhibitors such as AZD6244 are in clinical trials for the treatment of multiple cancers, including breast cancer. Targeted kinase inhibition can induce compensatory kinome changes, rendering ...single therapeutic agents ineffective. To identify target proteins to be used in a combinatorial approach to inhibit tumor cell growth, we used a novel strategy that identified microRNAs (miRNAs) that synergized with AZD6244 to inhibit the viability of the claudin-low breast cancer cell line MDA-MB-231. Screening of a miRNA mimic library revealed the ability of miR-9-3p to significantly enhance AZD6244-induced extracellular signal-regulated kinase inhibition and growth arrest, while miR-9-3p had little effect on growth alone. Promoter methylation of mir-9 genes correlated with low expression of miR-9-3p in different breast cancer cell lines. Consistent with miR-9-3p having synthetic enhancer tumor suppressor characteristics, miR-9-3p expression in combination with MEK inhibitor caused a sustained loss of c-MYC expression and growth inhibition. The beta 1 integrin gene (ITGB1) was identified as a new miR-9-3p target, and the growth inhibition seen with small interfering RNA knockdown or antibody blocking of ITGB1 in combination with MEK inhibitor phenocopied the growth inhibition seen with miR-9-3p plus AZD6244. The miRNA screen led to identification of a druggable protein, ITGB1, whose functional inhibition synergizes with MEK inhibitor.
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