Klaus P. Hoeflich

Calmodulin in Action: Diversity in Target Recognition and Activation Mechanisms

Recent structural studies on calmodulin complexes with anthrax adenylyl cyclase and rat Ca2+-activated K+ channel have uncovered unexpected ways by which calmodulin interacts with target proteins.

MinireviewCalmodulin in Action: Diversity in Target Recognition and Activation Mechanisms

Recent structural studies on calmodulin complexes with anthrax adenylyl cyclase and rat Ca2+-activated K+ channel have uncovered unexpected ways by which calmodulin interacts with target proteins.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

Mediation of TNF receptor-associated factor effector functions by apoptosis signal-regulating kinase-1 (ASK1)

Tumor necrosis factor-α (TNF), a major inflammatory cytokine, generates a wide variety of cellular responses via key cytoplasmic adaptor molecules named TNF receptor-associated factors (TRAFs). We report that TRAF2, TRAF5 and TRAF6 associate with apoptosis signal-regulating kinase 1 (ASK1), and a catalytically-inactive ASK1 mutant blocks stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase (JNK) activation by these TRAFs. A truncated derivative of TRAF2, which inhibits SAPK activation by TNF, blocks TNF-induced ASK1 activation. Furthermore, protection from TNF-induced cell death conferred by an ASK1 mutant is dependent upon TRAF2. Hence, ASK1 is a common mediator of TRAF-regulated SAPK and apoptosis signaling, and the TRAF2 – ASK1 connection completes the signaling cascade from TNF to SAPK/JNK activation.

Bcl-2 targeted to the endoplasmic reticulum can inhibit apoptosis induced by Myc but not etoposide in Rat-1 fibroblasts.

Bcl-2 is a key inhibitor of a broad range of apoptotic pathways, yet neither the mechanism of action nor the role of Bcl-2 subcellular localization are well understood. The subcellular localization of Bcl-2 includes the mitochondrial membrane as well as the contiguous membrane of the endoplasmic reticulum and nuclear envelope. Most studies suggest that the ability of Bcl-2 to confer cell survival is dependent upon its localization to the mitochondria. In this manuscript, we show that Bcl-2 targeted to the endoplasmic reticulum can inhibit Myc-, but not etoposide-induced apoptosis in the Rat-1 fibroblast cell line. By contrast, wild type Bcl-2 can inhibit apoptosis triggered by either death agonist. We further show both Myc and etoposide trigger disruption of mitochondrial membrane potential (MMP) and induce poly-ADP ribose polymerase (PARP) cleavage, but release of calcium was not evident. Bcl-2 abrogates apoptosis at or upstream of MMP depletion showing that Bcl-2 does not have to reside at the mitochondria to prevent apoptosis. These results further elucidate the biochemical events associated with Myc- and etoposide-induced apoptosis and significantly advance our understanding of Bcl-2 function.

Systems-wide analysis of K-Ras, Cdc42 and PAK4 signaling by quantitative phosphoproteomics

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.

Back Pocket Flexibility Provides Group II p21-Activated Kinase (PAK) Selectivity for Type I 1/2 Kinase Inhibitors.

Structure-based methods were used to design a potent and highly selective group II p21-activated kinase (PAK) inhibitor with a novel binding mode, compound 17. Hydrophobic interactions within a lipophilic pocket past the methionine gatekeeper of group II PAKs approached by these type I 1/2 binders were found to be important for improving potency. A structure-based hypothesis and strategy for achieving selectivity over group I PAKs, and the broad kinome, based on unique flexibility of this lipophilic pocket, is presented. A concentration-dependent decrease in tumor cell migration and invasion in two triple-negative breast cancer cell lines was observed with compound 17.

Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition

Significance Growing evidence suggests that successful intervention in many human cancers will require combinations of therapeutic agents. Critical to this effort will be a detailed understanding of the crosstalk between signaling networks that modulate proliferation, cell death, drug sensitivity, and acquired resistance. Here we investigated DNA-damage signaling elicited by small-molecule inhibitors against MAP/ERK kinase (MEK) and PI3K in melanoma cells. This work, performed using cutting-edge mass spectrometry proteomics, uncovered a burst of signaling among proteins in the DNA-damage pathway upon initiation of the cell-death program by agents targeting the RAS–RAF–MEK and PI3K–AKT–mTOR pathways. These signals may prove important to the short- and long-term sensitivity of tumor cells to MEK- and PI3K-targeted therapies. Targeted therapeutics that block signal transduction through the RAS–RAF–MEK and PI3K–AKT–mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941–mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.

The PI3K inhibitor taselisib overcomes letrozole resistance in a breast cancer model expressing aromatase.

Letrozole is a commonly used treatment option for metastatic hormone receptor-positive (HR+) breast cancer, but many patients ultimately relapse. Due to the importance of phosphoinositide-3 kinase (PI3K) in breast cancer, PI3K inhibitors such as taselisib are attractive for combination with endocrine therapies such as letrozole. Taselisib was evaluated as a single agent and in combination with letrozole in a breast cancer cell line engineered to express aromatase. The combination of taselisib and letrozole decreased cellular viability and increased apoptosis relative to either single agent. Signaling cross-talk between the PI3K and ER pathways was associated with efficacy for the combination. In a secreted factor screen, multiple soluble factors, including members of the epidermal and fibroblast growth factor families, rendered breast cancer cells non-responsive to letrozole. It was discovered that many of these factors signal through the PI3K pathway and cells remained sensitive to taselisib in the presence of the soluble factors. We also found that letrozole resistant lines have elevated PI3K pathway signaling due to an increased level of p110α, but are still sensitive to taselisib. These data provide rationale for clinical evaluation of PI3K inhibitors to overcome resistance to endocrine therapies in ER+ breast cancer.

Chapter 13 Mitogen-activated protein kinases and stress

Publisher Summary The mitogen-activated protein kinase (MAPK) superfamily plays an important role in transducing signals from the cell surface to the nucleus, effecting both the cells ability to cope with outside changes as well as cellular coordination in the case of multicellular organisms. The term “MAPK” is most widely used as a general denominator of this family of protein kinases. The MAPK acronym originally described the “microtubule-associated protein-2 kinase” but evolved into mitogen-activated protein kinase when it was discovered that the enzyme was induced by a variety of hormones and mitogens. Upon the molecular cloning of these enzymes, it was realized that they existed in several classes that were structurally related but distinctly regulated. MAPK is now commonly used to denote the entire class of protein-serine kinases that share the following features: the core functional unit of a MAPK module consists of a triad of three kinases that act sequentially, where MAPKs are activated via phosphorylation on both a threonine and tyrosine residue by selective upstream regulatory kinases, MAPK kinases (MAPKKs or MAP2Ks).