The paradox-breaking panRAF plus SRC family kinase inhibitor, CCT3833, is effective in mutant KRAS-driven cancers.

BACKGROUND: KRAS is mutated in ∼90% of pancreatic ductal adenocarcinomas, ∼35% of colorectal cancers and ∼20% of non-small-cell lung cancers. There has been recent progress in targeting G12CKRAS specifically, but therapeutic options for other mutant forms of KRAS are limited, largely because the complexity of downstream signaling and feedback mechanisms mean that targeting individual pathway components is ineffective. DESIGN: The protein kinases RAF and SRC are validated therapeutic targets in KRAS-mutant pancreatic ductal adenocarcinomas, colorectal cancers and non-small-cell lung cancers and we show that both must be inhibited to block growth of these cancers. We describe CCT3833, a new drug that inhibits both RAF and SRC, which may be effective in KRAS-mutant cancers. RESULTS: We show that CCT3833 inhibits RAF and SRC in KRAS-mutant tumors in vitro and in vivo, and that it inhibits tumor growth at well-tolerated doses in mice. CCT3833 has been evaluated in a phase I clinical trial (NCT02437227) and we report here that it significantly prolongs progression-free survival of a patient with a G12VKRAS spindle cell sarcoma who did not respond to a multikinase inhibitor and therefore had limited treatment options. CONCLUSIONS: New drug CCT3833 elicits significant preclinical therapeutic efficacy in KRAS-mutant colorectal, lung and pancreatic tumor xenografts, demonstrating a treatment option for several areas of unmet clinical need. Based on these preclinical data and the phase I clinical unconfirmed response in a patient with KRAS-mutant spindle cell sarcoma, CCT3833 requires further evaluation in patients with other KRAS-mutant cancers.

Prediction and monitoring of relapse in stage III melanoma using circulating tumor DNA.

BACKGROUND: The advent of effective adjuvant therapies for patients with resected melanoma has highlighted the need to stratify patients based on risk of relapse given the cost and toxicities associated with treatment. Here we assessed circulating tumor DNA (ctDNA) to predict and monitor relapse in resected stage III melanoma. PATIENTS AND METHODS: Somatic mutations were identified in 99/133 (74%) patients through tumor tissue sequencing. Personalized droplet digital PCR (ddPCR) assays were used to detect known mutations in 315 prospectively collected plasma samples from mutation-positive patients. External validation was performed in a prospective independent cohort (n = 29). RESULTS: ctDNA was detected in 37 of 99 (37%) individuals. In 81 patients who did not receive adjuvant therapy, 90% of patients with ctDNA detected at baseline and 100% of patients with ctDNA detected at the postoperative timepoint relapsed at a median follow up of 20 months. ctDNA detection predicted patients at high risk of relapse at baseline [relapse-free survival (RFS) hazard ratio (HR) 2.9; 95% confidence interval (CI) 1.5-5.6; P = 0.002] and postoperatively (HR 10; 95% CI 4.3-24; P < 0.001). ctDNA detection at baseline [HR 2.9; 95% CI 1.3-5.7; P = 0.003 and postoperatively (HR 11; 95% CI 4.3-27; P < 0.001] was also associated with inferior distant metastasis-free survival (DMFS). These findings were validated in the independent cohort. ctDNA detection remained an independent predictor of RFS and DMFS in multivariate analyses after adjustment for disease stage and BRAF mutation status. CONCLUSION: Baseline and postoperative ctDNA detection in two independent prospective cohorts identified stage III melanoma patients at highest risk of relapse and has potential to inform adjuvant therapy decisions.

Circulating tumor DNA predicts survival in patients with resected high-risk stage II/III melanoma.

Background: Patients with high-risk stage II/III resected melanoma commonly develop distant metastases. At present, we cannot differentiate between patients who will recur or those who are cured by surgery. We investigated if circulating tumor DNA (ctDNA) can predict relapse and survival in patients with resected melanoma. Patients and methods: We carried out droplet digital polymerase chain reaction to detect BRAF and NRAS mutations in plasma taken after surgery from 161 stage II/III high-risk melanoma patients enrolled in the AVAST-M adjuvant trial. Results: Mutant BRAF or NRAS ctDNA was detected (≥1 copy of mutant ctDNA) in 15/132 (11%) BRAF mutant patient samples and 4/29 (14%) NRAS mutant patient samples. Patients with detectable ctDNA had a decreased disease-free interval [DFI; hazard ratio (HR) 3.12; 95% confidence interval (CI) 1.79-5.47; P < 0.0001] and distant metastasis-free interval (DMFI; HR 3.22; 95% CI 1.80-5.79; P < 0.0001) versus those with undetectable ctDNA. Detectable ctDNA remained a significant predictor after adjustment for performance status and disease stage (DFI: HR 3.26, 95% CI 1.83-5.83, P < 0.0001; DMFI: HR 3.45, 95% CI 1.88-6.34, P < 0.0001). Five-year overall survival rate for patients with detectable ctDNA was 33% (95% CI 14%-55%) versus 65% (95% CI 56%-72%) for those with undetectable ctDNA. Overall survival was significantly worse for patients with detectable ctDNA (HR 2.63; 95% CI 1.40-4.96); P = 0.003) and remained significant after adjustment for performance status (HR 2.50, 95% CI 1.32-4.74, P = 0.005). Conclusion: ctDNA predicts for relapse and survival in high-risk resected melanoma and could aid selection of patients for adjuvant therapy. Clinical trial number: ISRCTN 81261306.

The European Society for Medical Oncology (ESMO) Precision Medicine Glossary.

Background: Precision medicine is rapidly evolving within the field of oncology and has brought many new concepts and terminologies that are often poorly defined when first introduced, which may subsequently lead to miscommunication within the oncology community. The European Society for Medical Oncology (ESMO) recognises these challenges and is committed to support the adoption of precision medicine in oncology. To add clarity to the language used by oncologists and basic scientists within the context of precision medicine, the ESMO Translational Research and Personalised Medicine Working Group has developed a standardised glossary of relevant terms. Materials and methods: Relevant terms for inclusion in the glossary were identified via an ESMO member survey conducted in Autumn 2016, and by the ESMO Translational Research and Personalised Medicine Working Group members. Each term was defined by experts in the field, discussed and, if necessary, modified by the Working Group before reaching consensus approval. A literature search was carried out to determine which of the terms, 'precision medicine' and 'personalised medicine', is most appropriate to describe this field. Results: A total of 43 terms are included in the glossary, grouped into five main themes-(i) mechanisms of decision, (ii) characteristics of molecular alterations, (iii) tumour characteristics, (iv) clinical trials and statistics and (v) new research tools. The glossary classes 'precision medicine' or 'personalised medicine' as technically interchangeable but the term 'precision medicine' is favoured as it more accurately reflects the highly precise nature of new technologies that permit base pair resolution dissection of cancer genomes and is less likely to be misinterpreted. Conclusions: The ESMO Precision Medicine Glossary provides a resource to facilitate consistent communication in this field by clarifying and raising awareness of the language employed in cancer research and oncology practice. The glossary will be a dynamic entity, undergoing expansion and refinement over the coming years.

Consensus on precision medicine for metastatic cancers: a report from the MAP conference.

Recent advances in biotechnologies have led to the development of multiplex genomic and proteomic analyses for clinical use. Nevertheless, guidelines are currently lacking to determine which molecular assays should be implemented in metastatic cancers. The first MAP conference was dedicated to exploring the use of genomics to better select therapies in the treatment of metastatic cancers. Sixteen consensus items were covered. There was a consensus that new technologies like next-generation sequencing of tumors and ddPCR on circulating free DNA have convincing analytical validity. Further work needs to be undertaken to establish the clinical utility of liquid biopsies and the added clinical value of expanding from individual gene tests into large gene panels. Experts agreed that standardized bioinformatics methods for biological interpretation of genomic data are needed and that precision medicine trials should be stratified based on the level of evidence available for the genomic alterations identified.

Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA.

BACKGROUND: The application of precision medicine in oncology requires in-depth characterisation of a patient's tumours and the dynamics of their responses to treatment. PATIENTS AND METHODS: We used next-generation sequencing of circulating cell-free DNA (cfDNA) to monitor the response of a KIT p.L576P-mutant metastatic vaginal mucosal melanoma to sequential targeted, immuno- and chemotherapy. RESULTS: Despite a KIT mutation, the response to imatinib was mixed. Unfortunately, tumours were not accessible for molecular analysis. To study the mechanism underlying the mixed clinical response, we carried out whole-exome sequencing and targeted longitudinal analysis of cfDNA. This revealed two tumour subclones; one with a KIT mutation that responded to imatinib and a second KIT-wild-type subclone that did not respond to imatinib. Notably, the subclones also responded differently to immunotherapy. However, both subclones responded to carboplatin/paclitaxel, and although the KIT-wild-type subclone progressed after chemotherapy, it responded to subsequent re-administration of paclitaxel. CONCLUSION: We show that cfDNA can reveal tumour evolution and subclonal responses to therapy even when biopsies are not available.

A personalised medicine approach for ponatinib-resistant chronic myeloid leukaemia.

BACKGROUND: Chronic myeloid leukaemia (CML) is characterised by the presence of a fusion driver oncogene, BCR-ABL1, which is a constitutive tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are the central treatment strategy for CML patients and have significantly improved survival rates, but the T315I mutation in the kinase domain of BCR-ABL1 confers resistance to all clinically approved TKIs, except ponatinib. However, compound mutations can mediate resistance even to ponatinib and remain a clinical challenge in CML therapy. Here, we investigated a ponatinib-resistant CML patient through whole-genome sequencing (WGS) to identify the cause of resistance and to find alternative therapeutic targets. PATIENTS AND METHODS: We carried out WGS on a ponatinib-resistant CML patient and demonstrated an effective combination therapy against the primary CML cells derived from this patient in vitro. RESULTS: Our findings demonstrate the emergence of compound mutations in the BCR-ABL1 kinase domain following ponatinib treatment, and chromosomal structural variation data predicted amplification of BCL2. The primary CD34(+) CML cells from this patient showed increased sensitivity to the combination of ponatinib and ABT-263, a BCL2 inhibitor with a negligible effect against the normal CD34(+) cells. CONCLUSION: Our results show the potential of personalised medicine approaches in TKI-resistant CML patients and provide a strategy that could improve clinical outcomes for these patients.

Whole-genome sequencing reveals complex mechanisms of intrinsic resistance to BRAF inhibition.

BACKGROUND: BRAF is mutated in ∼42% of human melanomas (COSMIC. http://www.sanger.ac.uk/genetics/CGP/cosmic/) and pharmacological BRAF inhibitors such as vemurafenib and dabrafenib achieve dramatic responses in patients whose tumours harbour BRAF(V600) mutations. Objective responses occur in ∼50% of patients and disease stabilisation in a further ∼30%, but ∼20% of patients present primary or innate resistance and do not respond. Here, we investigated the underlying cause of treatment failure in a patient with BRAF mutant melanoma who presented primary resistance. METHODS: We carried out whole-genome sequencing and single nucleotide polymorphism (SNP) array analysis of five metastatic tumours from the patient. We validated mechanisms of resistance in a cell line derived from the patient's tumour. RESULTS: We observed that the majority of the single-nucleotide variants identified were shared across all tumour sites, but also saw site-specific copy-number alterations in discrete cell populations at different sites. We found that two ubiquitous mutations mediated resistance to BRAF inhibition in these tumours. A mutation in GNAQ sustained mitogen-activated protein kinase (MAPK) signalling, whereas a mutation in PTEN activated the PI3 K/AKT pathway. Inhibition of both pathways synergised to block the growth of the cells. CONCLUSIONS: Our analyses show that the five metastases arose from a common progenitor and acquired additional alterations after disease dissemination. We demonstrate that a distinct combination of mutations mediated primary resistance to BRAF inhibition in this patient. These mutations were present in all five tumours and in a tumour sample taken before BRAF inhibitor treatment was administered. Inhibition of both pathways was required to block tumour cell growth, suggesting that combined targeting of these pathways could have been a valid therapeutic approach for this patient.

MiR-378a inhibits glucose metabolism by suppressing GLUT1 in prostate cancer.

Prostate cancer (PCa) is the fifth leading cause of cancer related deaths worldwide, in part due to a lack of molecular stratification tools that can distinguish primary tumours that will remain indolent from those that will metastasise. Amongst potential molecular biomarkers, microRNAs (miRs) have attracted particular interest because of their high stability in body fluids and fixed tissues. These small non-coding RNAs modulate several physiological and pathological processes, including cancer progression. Herein we explore the prognostic potential and the functional role of miRs in localised PCa and their relation to nodal metastasis. We define a 7-miR signature that is associated with poor survival independently of age, Gleason score, pathological T state, N stage and surgical margin status and that is also prognostic for disease-free survival in patients with intermediate-risk localised disease. Within our 7-miR signature, we show that miR-378a-3p (hereafter miR-378a) levels are low in primary tumours compared to benign prostate tissue, and also lower in Gleason score 8-9 compared to Gleason 6-7 PCa. We demonstrate that miR-378a impairs glucose metabolism and reduces proliferation in PCa cells through independent mechanisms, and we identify glucose transporter 1 (GLUT1) messenger RNA as a direct target of miR-378a. We show that GLUT1 inhibition hampers glycolysis, leading to cell death. Our data provides a rational for a new PCa stratification strategy based on miR expression, and it reveals that miR-378a and GLUT1 are potential therapeutic targets in highly aggressive glycolytic PCa.

Sorafenib and dacarbazine as first-line therapy for advanced melanoma: phase I and open-label phase II studies.

METHOD: The safety of oral sorafenib up to a maximum protocol-specified dose combined with dacarbazine in patients with metastatic, histologically confirmed melanoma was investigated in a phase I dose-escalation study and the activity of the combination was explored in an open-label phase II study. RESULTS: In the phase I study, three patients were treated with sorafenib 200 mg twice daily (b.i.d.) plus 1000 mg m(-2) dacarbazine on day 1 of a 21-day cycle and 15 patients had the sorafenib dose escalated to 400 mg b.i.d. without reaching the maximum tolerated dose of the combination. In the phase II study (n=83), the overall response rate was 12% (95% CI: 6, 21): one complete and nine partial, with median response duration of 46.7 weeks. Stable disease was the best response in 37%; median duration was 13.3 weeks. Median overall survival (OS) was 37.0 weeks (95% CI: 33.9, 46.0). CONCLUSION: Oral sorafenib combined with dacarbazine had acceptable toxicity and some antineoplastic activity against metastatic melanoma.

Symptomatic hepatothorax presenting 25 years after penetrating thoracoabdominal injury.

Hepatic herniation through the diaphragm is a rare finding. It generally occurs due to a congenital diaphragmatic abnormality or blunt trauma resulting in a diaphragmatic defect. Making the diagnosis is difficult, as there are few definitive clinical signs and chest radiograph (CXR) findings may be non-specific. To our knowledge, only a single case report exists of penetrating right diaphragm injury leading to hepatic herniation. A 42-year-old man presented to the emergency department of a regional hospital with hyperglycaemia and exertional dyspnoea. He was diagnosed with diabetes mellitus type 2. He gave a history of smoking for 15 pack-years, was negative for retroviral disease and had no history of pulmonary tuberculosis. He had no significant surgical history but reported being stabbed with a knife in 1995. The point of entry was below the level of the nipple in the right anterior axillary line. At the time, he was treated with an intercostal drain and discharged home. CXR showed a right-sided chest mass. We considered a differential diagnosis of pulmonary consolidation, diaphragm eventration or hepatothorax. Computerized tomography of the chest and abdomen demonstrated apparent intrathoracic extension of the right liver lobe and partial attenuation of the superior vena cava and right atrium due to a mass effect. The upper border of the liver abutted the aortic arch. Surgical treatment options were discussed. The patient declined surgery and will be followed up as an outpatient.

Importance of low affinity Elf-1 sites in the regulation of lymphoid-specific inducible gene expression.

Elf-1 is an Ets family transcription factor that regulates a number of inducible lymphoid-specific genes, including those encoding interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and the IL-2 receptor (IL-2R) alpha chain. A minimal oligonucleotide spanning the IL-2R alpha Elf-1 site (-97/-84) bound Elf-1 poorly, but binding activity markedly increased when this oligonucleotide was multimerized or flanking sequences were added. This result is consistent with the requirement of accessory proteins for efficient Elf-1 binding, as has been demonstrated for the GM-CSF and IL-3 promoters. A binding site selection analysis revealed the optimal Elf-1 consensus motif to be A(A/t)(C/a)CCGGAAGT(A/S), which is similar to the consensus motif for the related Drosophila E74 protein. This minimal high affinity site could bind Elf-1 and functioned as a stronger transcription element than the -97/-84 IL-2R alpha oligonucleotide when cloned upstream of a heterologous promoter. In contrast, in the context of the IL-2R alpha promoter, conversion of the naturally occurring low affinity Elf-1 site to an optimal site decreased inducible activation of a reporter construct in Jurkat cells. This finding may be explained by the observation that another Ets family protein, ER GB/Fli-1, can efficiently bind only to the optimal site, and in this context, interferes with Elf-1 binding. Therefore, high affinity Elf-1 sites may lack sufficient binding specificity, whereas naturally occurring low affinity sites presumably favor the association of Elf-1 in the context of accessory proteins. These findings offer an explanation for the lack of optimal sites in any of the known Elf-1-regulated genes.

Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C.

Receptor tyrosine kinase-mediated activation of the Raf-1 protein kinase is coupled to the small guanosine triphosphate (GTP)-binding protein Ras. By contrast, protein kinase C (PKC)-mediated activation of Raf-1 is thought to be Ras independent. Nevertheless, stimulation of PKC in COS cells led to activation of Ras and formation of Ras-Raf-1 complexes containing active Raf-1. Raf-1 mutations that prevent its association with Ras blocked activation of Raf-1 by PKC. However, the activation of Raf-1 by PKC was not blocked by dominant negative Ras, indicating that PKC activates Ras by a mechanism distinct from that initiated by activation of receptor tyrosine kinases.

Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients.

INTRODUCTION: Tumour burden is a prognostic biomarker in metastatic melanoma. However, tumour burden is difficult to measure and there are currently no reliable surrogate biomarkers to easily and reliably determine it. The aim of this study was to assess the potential of plasma total cell free DNA as biomarker of tumour burden and prognosis in metastatic melanoma patients. MATERIALS AND METHODS: A prospective biomarker cohort study for total plasma circulating cell-free DNA (cfDNA) concentration was performed in 43 metastatic melanoma patients. For 38 patients, paired blood collections and scan assessments were available before treatment and at first response evaluation. Tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients. RESULTS: Baseline cfDNA concentration correlated with pre-treatment tumour burden (ρ = 0.52, P < 0.001). Baseline cfDNA levels correlated significantly with hazard of death and overall survival, and a cut off value of 89 pg/µl identified two distinct prognostic groups (HR = 2.22 for high cfDNA, P = 0.004). Patients with cfDNA ≥89 pg/µl had shorter OS (10.0 versus 22.7 months, P = 0.009; HR = 2.22 for high cfDNA, P = 0.004) and the significance was maintained when compared with lactic dehydrogenase (LDH) in a multivariate analysis. We also found a correlation between the changes of cfDNA and treatment-related changes in tumour burden (ρ = 0.49, P = 0.002). In addition, the ratio between baseline cfDNA and tumour burden was prognostic (HR = 2.7 for cfDNA/tumour volume ≥8 pg/(µl*cm3), P = 0.024). CONCLUSIONS: We have demonstrated that cfDNA is a surrogate marker of tumour burden in metastatic melanoma patients, and that it is prognostic for overall survival.

Phosphorylation and activation of B-Myb by cyclin A-Cdk2.

BACKGROUND: Cyclins and their catalytic partners, the cyclin-dependent kinases (Cdks), function as key regulators of the eukaryotic cell cycle. Specific cyclin-Cdk complexes are active at successive stages during the cell cycle and control cell-cycle progression by phosphorylating specific target proteins, most of which have not yet been identified. B-Myb, a conserved member of the Myb oncoprotein family, is a sequence-specific DNA-binding protein expressed in virtually all proliferating mammalian cells. Increasing evidence suggests that B-Myb plays an important role during the late G1 and early S phases of the cell cycle. In this study, we have examined the regulation of B-Myb activity by cyclin-Cdks. RESULTS: We found that the transcriptional transactivation potential of B-Myb was repressed by a regulatory domain located at the carboxyl terminus of the protein. Coexpression of B-Myb and cyclin A relieved this repression by phosphorylation of B-Myb in its carboxy-terminal region. Tryptic phosphopeptide mapping revealed that endogenous B-Myb was phosphorylated in cells undergoing S phase. CONCLUSIONS: This work provides evidence for a link between the Myb oncoprotein family and the cell-cycle machinery. We have shown that the carboxyl terminus of B-Myb acts as a cell-cycle sensor that regulates the transactivation function of B-Myb. Moreover, our studies have identified B-Myb as a target of cyclin A-Cdk2 and have indicated that B-Myb activity is regulated by phosphorylation mediated by cyclin A-Cdk2.

S338 phosphorylation of Raf-1 is independent of phosphatidylinositol 3-kinase and Pak3.

The Raf-1 serine/threonine protein kinase requires phosphorylation of the serine at position 338 (S338) for activation. Ras is required to recruit Raf-1 to the plasma membrane, which is where S338 phosphorylation occurs. The recent suggestion that Pak3 could stimulate Raf-1 activity by directly phosphorylating S338 through a Ras/phosphatidylinositol 3-kinase (Pl3-K)/-Cdc42-dependent pathway has attracted much attention. Using a phospho-specific antibody to S338, we have reexamined this model. Using LY294002 and wortmannin, inhibitors of Pl3-K, we find that growth factor-mediated S338 phosphorylation still occurs, even when Pl3-K activity is completely blocked. Although high concentrations of LY294002 and wortmannin did suppress S338 phosphorylation, they also suppressed Ras activation. Additionally, we show that Pak3 is not activated under conditions where S338 is phosphorylated, but when Pak3 is strongly activated, by coexpression with V12Cdc42 or by mutations that make it independent of Cdc42, it did stimulate S338 phosphorylation. However, this occurred in the cytosol and did not stimulate Raf-1 kinase activity. The inability of Pak3 to activate Raf-1 was not due to an inability to stimulate phosphorylation of the tyrosine at position 341 but may be due to its inability to recruit Raf-1 to the plasma membrane. Taken together, our data show that growth factor-stimulated Raf-1 activity is independent of Pl3-K activity and argue against Pak3 being a physiological mediator of S338 phosphorylation in growth factor-stimulated cells.

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes.

Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. We demonstrate here that this proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-alpha inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (alpha, beta, delta, epsilon, eta, and zeta) were employed. Transient transfection of these PKC mutants into Cos-7 cells showed that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes alpha and eta) are potent activators of c-Raf1, atypical PKC-zeta cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-alpha and PKC-eta was abrogated for RafCAAX, which is a membrane-localized, partially active form of c-Raf1. We further established that activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, we describe a novel Raf desensitization induced by PKC-alpha, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in 12-O-tetradecanoylphorbol-13-acetate induced mitogenesis and provide evidence for multiple PKC controls acting on this MAPK cascade.

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1.

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.

Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling.

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

Radioactive labeling of recombinant antibody fragments by phosphorylation using human casein kinase II and [gamma-32P]-ATP.

A wide range of antibody fragments can be expressed in bacteria and detected immunochemically via peptide tags. Using specially designed tags, we have developed a strategy for radiolabeling antibody fragments secreted from bacteria. Tagged antibody fragments were secreted either into the bacterial periplasm or the culture medium. The tag was not subject to proteolysis either in the broth or in human plasma. After affinity purification the antibody fragments were phosphorylated with [gamma-32P]ATP and casein kinase II. The labeled fragments were used in a gel band-shift assay to measure antigen binding affinities. In contrast to non site-specific methods such as radioiodination, antibodies labeled with casein kinase II retain full immunoreactivity. Radioactively phosphorylated antibody fragments may have many other applications, including radioimmunoassays and radioimmunotherapy.