Synthetic Lethal Interaction with BCL-XL Blockade Deepens Response to Cetuximab in Patient-Derived Models of Metastatic Colorectal Cancer.

PURPOSE: Approximately 20% of patients with RAS wild-type metastatic colorectal cancer (mCRC) experience objective responses to the anti-EGFR antibody cetuximab, but disease eradication is seldom achieved. The extent of tumor shrinkage correlates with long-term outcome. We aimed to find rational combinations that potentiate cetuximab efficacy by disrupting adaptive dependencies on antiapoptotic molecules (BCL2, BCL-XL, MCL1). EXPERIMENTAL DESIGN: Experiments were conducted in patient-derived xenografts (PDX) and organoids (PDXO). Apoptotic priming was analyzed by BH3 profiling. Proapoptotic and antiapoptotic protein complexes were evaluated by co-immunoprecipitation and electroluminescence sandwich assays. The effect of combination therapies was assessed by caspase activation in PDXOs and by monitoring PDX growth. RESULTS: A population trial in 314 PDX cohorts, established from as many patients, identified 46 models (14.6%) with appreciable (>50% tumor shrinkage) but incomplete response to cetuximab. From these models, 14 PDXOs were derived. Cetuximab primed cells for apoptosis, but only concomitant blockade of BCL-XL precipitated cell death. Mechanistically, exposure to cetuximab induced upregulation of the proapoptotic protein BIM and its sequestration by BCL-XL. Inhibition of BCL-XL resulted in displacement of BIM, which was not buffered by MCL1 and thereby became competent to induce apoptosis. In five PDX models, combination of cetuximab and a selective BCL-XL inhibitor triggered apoptosis and led to more pronounced tumor regressions and longer time to relapse after treatment discontinuation than cetuximab alone. CONCLUSIONS: In mCRC tumors that respond to cetuximab, antibody treatment confers a synthetic-lethal dependency on BCL-XL. Targeting this dependency unleashes apoptosis and increases the depth of response to cetuximab.

Effective drug combinations in breast, colon and pancreatic cancer cells.

Combinations of anti-cancer drugs can overcome resistance and provide new treatments1,2. The number of possible drug combinations vastly exceeds what could be tested clinically. Efforts to systematically identify active combinations and the tissues and molecular contexts in which they are most effective could accelerate the development of combination treatments. Here we evaluate the potency and efficacy of 2,025 clinically relevant two-drug combinations, generating a dataset encompassing 125 molecularly characterized breast, colorectal and pancreatic cancer cell lines. We show that synergy between drugs is rare and highly context-dependent, and that combinations of targeted agents are most likely to be synergistic. We incorporate multi-omic molecular features to identify combination biomarkers and specify synergistic drug combinations and their active contexts, including in basal-like breast cancer, and microsatellite-stable or KRAS-mutant colon cancer. Our results show that irinotecan and CHEK1 inhibition have synergistic effects in microsatellite-stable or KRAS-TP53 double-mutant colon cancer cells, leading to apoptosis and suppression of tumour xenograft growth. This study identifies clinically relevant effective drug combinations in distinct molecular subpopulations and is a resource to guide rational efforts to develop combinatorial drug treatments.

Sustained Inhibition of HER3 and EGFR Is Necessary to Induce Regression of HER2-Amplified Gastrointestinal Carcinomas.

PURPOSE: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities. EXPERIMENTAL DESIGN: The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays. RESULTS: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib-or, importantly, monotherapy with afatinib-resulted in overt tumor shrinkage. CONCLUSIONS: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519-31. ©2015 AACR.

HER2 activating mutations are targets for colorectal cancer treatment.

UNLABELLED: The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. SIGNIFICANCE: HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer.

IGF2 is an actionable target that identifies a distinct subpopulation of colorectal cancer patients with marginal response to anti-EGFR therapies.

Among patients with colorectal cancer who benefit from therapy targeted to the epidermal growth factor receptor (EGFR), stable disease (SD) occurs more frequently than massive regressions. Exploring the mechanisms of this incomplete sensitivity to devise more efficacious treatments will likely improve patients' outcomes. We tested therapies tailored around hypothesis-generating molecular features in patient-derived xenografts ("xenopatients"), which originated from 125 independent samples that did not harbor established resistance-conferring mutations. Samples from xenopatients that responded to cetuximab, an anti-EGFR agent, with disease stabilization displayed high levels of EGFR family ligands and receptors, indicating high EGFR pathway activity. Five of 21 SD models (23.8%) characterized by particularly high expression of EGFR and EGFR family members regressed after intensified EGFR blockade by cetuximab and a small-molecule inhibitor. In addition, a subset of cases in which enhanced EGFR inhibition was unproductive (6 of 16, 37.5%) exhibited marked overexpression of insulin-like growth factor 2 (IGF2). Enrichment of IGF2 overexpressors among cases with SD was demonstrated in the entire xenopatient collection and was confirmed in patients by mining clinical gene expression data sets. In functional studies, IGF2 overproduction attenuated the efficacy of cetuximab. Conversely, interception of IGF2-dependent signaling in IGF2-overexpressing xenopatients potentiated the effects of cetuximab. The clinical implementation of IGF inhibitors awaits reliable predictors of response, but the results of this study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker of reduced tumor sensitivity to anti-EGFR therapy and a determinant of response to combined IGF2/EGFR targeting.

Primary and acquired resistance to EGFR-targeted therapies in colorectal cancer: impact on future treatment strategies.

Only approximately 10% of genetically unselected patients with chemorefractory metastatic colorectal cancer experience tumor regression when treated with the anti-epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab ("primary" or "de novo" resistance). Moreover, nearly all patients whose tumors initially respond inevitably become refractory ("secondary" or "acquired" resistance). An ever-increasing number of predictors of both primary and acquired resistance to anti-EGFR antibodies have been described, and it is now evident that most of the underlying mechanisms significantly overlap. By trying to extrapolate a unifying perspective out of many idiosyncratic details, here, we discuss the molecular underpinnings of therapeutic resistance, summarize research efforts aimed to improve patient selection, and present alternative therapeutic strategies that are now under development to increase response and combat relapse.

Gonadal and uterine function in female survivors treated by chemotherapy, radiotherapy, and/or bone marrow transplantation for childhood malignant and non-malignant diseases.

OBJECTIVE: To evaluate gonadal function and uterine volume in a cohort of female survivors treated by chemotherapy, radiotherapy, and/or stem cell transplantation (SCT) for childhood malignant and non-malignant diseases. DESIGN: An observational study. SETTING: S. Matteo Hospital, Pavia, Italy. POPULATION: A cohort of 135 female survivors. METHODS: A clinical, hormonal, and ultrasonographic evaluation. Thirty-three patients (24%) had non-malignant haematologic diseases (thalassaemia or sickle cell anaemia), 68 (50%) had leukaemia, 23 (17%) had lymphomas, and 11 (8%) had solid tumours. In total, 106 patients had received SCT, preceded by a conditioning regimen. MAIN OUTCOME MEASURES: Anti-Müllerian hormone (AMH) and Inhibin-B, and uterine volume. RESULTS: The median concentrations of AMH and Inhibin-B in the entire cohort were 0.12 ng/ml (interquartile range, IQR, 0.1-0.5 ng/ml) and 3.5 pg/ml (IQR 0.1-13.2 pg/ml), respectively. In a stepwise ordered logistic regression analysis, conventional chemotherapy for the treatment of malignancies, as opposed to total body irradiation (TBI), was the only oncologically significant predictor of increased AMH levels (OR 4.8, 95% CI 1.9-12, P < 0.001). Conditioning treatment before or after menarche did not influence AMH concentrations (P = 0.24). The best predictor of reduced uterine volume was TBI during the preparation for the allograft (OR 3.5, 95% CI 1.4-8.4, P = 0.006). Increasing age at treatment (OR 0.86, 95% CI 0.77-0.95, P = 0.04), chemotherapy, as opposed to other treatments (OR 0.09, 95% CI 0.03-0.28, P < 0.001), and solid tumours as opposed to either leukaemia/lymphomas or non-malignant diseases (OR 0.2, 95% CI 0.07-0.56, P = 0.002) were associated with larger uterine volumes. CONCLUSIONS: Conditioning therapies for SCT, including TBI, had the worst effects on uterine volume and gonadal reserve. Increasing age at treatment and conventional chemotherapy were associated with less detrimental effects on uterine volume.

Effects of uncomplicated vaginal delivery and epidural analgesia on fetal arterial acid-base parameters at birth in gestational diabetes.

AIM: To investigate the effects of uncomplicated vaginal delivery and epidural analgesia on fetal acid-base parameters in women with gestational diabetes (GDM) compared with controls. METHODS: A retrospective case-control study of 142 women with gestational diabetes and 284 controls. To evaluate the effect of diabetes and analgesia on acid-base status correcting for potential confounders we used ordered logistic equations including quartiles of fetal arterial acid-base parameters collected at birth as outcomes and categories of diabetes and epidural analgesia as explanatory variables. RESULTS: In the GDM group cord base deficit (-2.63 mmol/l, interquartile range [IQR]=4.2 to -0.65 mmol/l vs. -1.9 mmol/l, IQR=-3.3 to -0.2 mmol/l, p=0.009, odds ratio (OR)=1.51, 95% confidence interval (CI)=1.04-2.18) was lower and concentration of calcium higher (1.49 mmol/l, IQR=1.42-1.56 mmol/l vs. 1.47 mmol/l, IQR=1.41-1.51 mmol/l, p=0.009, OR=1.69, 95% CI=1.12-2.56) compared with controls. Epidural analgesia in the GDM group was associated with reduced cord concentration of glucose (84.0mg/dl [4.7 mmol/l], IQR=70-103.3mg/dl vs. 92.5mg/dl [5.1 mmol/l], IQR=76.5-121.8 mg/dl, p=0.004), lactate (2.65 mmol/l (IQR=1.80-4.20) vs. 3.70 mmol/l (IQR=2.90-5.55 mmol/l), p=0.002) and less pronounced base deficit (-2.05 mmol/l, IQR=-3.90 to -0.17 mmol/l vs. -2.8, IQR=-5.57 to -1.05 mmol/l, p=0.01, OR=0.7, 95% CI=0.49-0.99). CONCLUSIONS: In uncomplicated pregnancies and deliveries, well-controlled gestational diabetes mellitus has potentially significant detrimental effects on fetal acid-base status at birth. Epidural analgesia reduces cord arterial glucose and lactates.

Model for total evaluation of a phytosanitary intervention in the context of new methodology of <>.

The model of the "GREAT FILIERA" is proposed for as it concerns the total evaluation of a phytosanitary intervention in the vine-growing and wine-producing sector. This model is essentially based on the application of a new methodology used for the evaluation of a control system (Carbonneau & Cargnello, 2003) already tested in applicatory phase. The total evaluation is determined by an appropriate panel of judges. They have considered and assigned a "weigh" in % in one scale of priority (number that follows the variable) and beyond fifty descriptors of the productive system, constituting the total system.

Pattern of monosynaptic heteronymous Ia connections in the human lower limb.

Changes in the firing probability of single motor units in response to electrical stimulation of muscle nerves were used to derive the projections of muscle spindle Ia afferents to the motoneurones of various leg and thigh muscles. Discharges of units in soleus, gastrocnemius medialis, peroneus brevis, tibialis anterior, quadriceps, biceps femoris and semitendinosus were investigated after stimulation of inferior soleus, gastrocnemius medialis, superficial peroneal, deep peroneal and femoral nerves. Homonymous facilitation, occurring at the same latency as the H reflex and therefore attributed to monosynaptic Ia EPSPs, was found in virtually all the sampled units. In many motor nuclei an early facilitation was also evoked by heteronymous low-threshold afferents. The heteronymous facilitation was considered to be mediated through a monosynaptic pathway when the difference between the central latencies of heteronymous and homonymous peaks was not more than 0.2 ms. The heteronymous Ia connections were widely distributed. In particular, monosynaptic coupling between muscles operating at different joints appears to be the rule in humans, though it is rare between ankle and knee muscles in the cat and the baboon.

A study on apoenzyme from Rhodotorula gracilis D-amino acid oxidase.

The apoenzyme of D-amino acid oxidase from Rhodotorula gracilis was obtained at pH 7.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M potassium phosphate, 0.3 mM EDTA, 5 mM 2-mercaptoethanol and 20% glycerol. To recover a reconstitutable and highly stable apoprotein, it is essential that phosphate ions and glycerol be present at high concentrations. Apo-D-amino acid oxidase is entirely present as a monomeric protein, while the reconstituted holoenzyme is a dimer of 79 kDa. The equilibrium binding of FAD to apoprotein was measured from the quenching of flavin fluorescence and by differential spectroscopy: a Kd of 2.0 x 10(-8) M was calculated. The kinetics of formation of the apoprotein-FAD complex were studied by the quenching of protein and flavin fluorescence, by differential spectroscopy and by activity measurements. In all cases a two-stage process was shown to be present with a fairly rapid first phase, followed by a slow secondary change which represents only 4-6% of the total recombination process. In no conditions was a lag in the recovery of maximum catalytic activity observed. The process of FAD binding to yeast D-amino acid oxidase appears to be of the type Apo + FAD in equilibrium holoenzyme, even though the existence of a transient intermediate not detectable under our conditions cannot be ruled out.

Bereitschaftspotential in a simple movement or in a motor sequence starting with the same simple movement.

The Bereitschaftspotential (BP) recorded from 3 derivations (vertex, left and right precentral areas) in 20 right-handed, normal young subjects was compared in 2 kinds of motor task: a simple movement (task A) and a motor sequence (task B) starting with the simple movement (A). Differences in the onset time and amplitude of the BP were observed: the onset was earlier and the amplitude was larger in the sequential motor task (B) than in the simple one (A). These differences were more important at the vertex (Cz) and in the right precentral area (C4) than in the left contralateral precentral area (C3). These results suggest that the preparatory processes involved in a motor sequence do not exclusively concern the initial movement but also the remainder of the motor task and that the BP is dependent upon the duration or the complexity of the motor task to be executed. The BP seems on temporal grounds to be a global and not a partial expression of a motor task. The changes in the onset time and amplitude of the BP are maximal at the vertex and this could be related to a greater and perhaps earlier activation of SMA in complex sequential motor tasks.

Immunochemical studies on Rhodotorula gracilis D-amino acid oxidase.

Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified from R. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.

Pattern of propriospinal-like excitation to different species of human upper limb motoneurones.

1. The pattern of distribution of non-monosynaptic (propriospinal-like) excitation to various motor nuclei (deltoid, extensors and flexors of the elbow, the wrist and the fingers) was investigated. 2. Changes in the firing probability of individual voluntarily activated motor units were studied following conditioning stimuli. Conditioning volleys were evoked by weak electrical stimuli applied to various mixed nerves (circumflex, musculocutaneous, median, radial, ulnar) and to the skin. 3. In all investigated nuclei stimulation of the 'homonymous' nerve evoked a peak of increased firing probability with a latency which was 2-7 ms longer than the monosynaptic Ia latency. The average central delay of the late excitation, measured from monosynaptic latency, seems to depend only on the segmental level of the motor nucleus: the more caudal the nucleus the longer the latency. This strongly suggest a transmission through neurones located above the cervical enlargement, as are C3--C4 propriospinal neurones in the cat. 4. Both group I muscle and cutaneous afferents were shown to contribute to propriospinal-like excitation. It is argued that a spatial facilitation of the effects evoked by these two inputs might explain why the threshold of late excitation is always below that of the monosynaptic Ia excitation in motoneurones. 5. The pattern of distribution of propriospinal-like excitation was diffuse: stimulation of each mixed nerve was able to evoke excitation in all investigated motor nuclei. Similarly, stimulation of a given skin field could produce excitation of biceps and wrist flexor and extensor units. 6. Each motor nucleus therefore receives excitation from a multimodal and wide range peripheral input. However, it is argued that what appears as a diffuse pattern might simply reflect connections which are not used in each movement but appropriately selected by higher centres.

Abnormal ocular movements in Parkinson's disease. Evidence for involvement of dopaminergic systems.

Quantitated automated electro-oculographic data from 45 parkinsonian patients were compared with those from 30 normal control subjects. Patients were selected with idiopathic Parkinson's disease without other associated neurological disease or dementia; 20 had never received antiparkinsonian drugs and in 25 such treatment had been stopped for at least 2 days. Saccade latency, amplitude and peak velocity, smooth pursuit peak velocity, optokinetic nystagmus (OKN) maximal and mean velocities and vestibulo-ocular reflex (VOR) suppression by vision or imagination were significantly altered in patients, whereas VOR gain in darkness was normal. Alterations of saccade latency and smooth pursuit peak velocity were more severe in the more advanced stages of the disease and saccade latency directed towards the symptomatic side was slightly delayed in hemiparkinsonian patients. Saccade amplitude improved 90 min after a single oral dose of L-DOPA. These results suggest a possible dopaminergic control of some ocular movements.

Properties of D-amino-acid oxidase from Rhodotorula gracilis.

The flavoprotein D-amino-acid oxidase was purified to homogeneity from the yeast Rhodotorula gracilis by a highly reproducible procedure. The amino acid composition of the protein was determined; the protein monomer had a molecular mass of 39 kDa and contained one molecule of FAD. The ratio between A274/A455 was about 8.2. D-Amino-acid oxidase from yeast showed typical flavin spectral perturbations on binding of the competitive inhibitor benzoate and was reduced by D-alanine under anaerobiosis. The enzyme reacted readily with sulfite to form a covalent reversible adduct and stabilized the red anionic form of the flavin semiquinone on photoreduction in the presence of 5-deazariboflavin; the 3,4-dihydro-FAD form was not detectable after reduction with sodium borohydride. Thus D-amino-acid oxidase from yeast exhibited most of the general properties of the dehydrogenase/oxidase class of flavoproteins; at the same time, the enzyme showed some peculiar features with respect to the same protein from pig kidney.

A new implemented version of program MIR (MIR II): analysis and identification of mathematical models in enzyme and transport kinetic studies.

The computer program MIR previously described (R. Bianchi, G.M. Hanozet and M. Pilone Simonetta, Comput. Prog. Biomed. 16 (1983) 189) that fits trial rate laws to enzyme and transport steady-state kinetic data by the least-squares method has been enhanced. The new version MIR II is an interactive program and it consists of five major routines and a larger number of smaller program elements to perform the linear (three different functional forms) and non-linear (eleven mathematical models) regression analysis of kinetic data from enzyme and transmembrane transport experiments, also in the presence of inhibitors. Other features of the new program include a set of statistics and tests useful for the model building process, for the development of the mathematical model and for its validation and maintenance. An algorithm for fitting a straight line taking into account errors in both x and y is also provided.

Two weeks of treatment with deprenyl (selegiline) does not prolong L-dopa effect in parkinsonian patients: a double-blind cross-over placebo-controlled trial.

We investigated the influence of 2 weeks of treatment with deprenyl on the acute effect of a single dose of 200 mg of L-dopa in a double-blind cross-over placebo-controlled trial in 16 parkinsonian patients with wearing-off phenomena. Deprenyl induced no significant benefit in the duration or the maximal improvement of L-dopa's effect. Moreover, deprenyl treatment did not improve the patient's motor status.

Phenylglyoxal modification of arginines in mammalian D-amino-acid oxidase.

The presence of arginine in the active center of D-amino-acid oxidase is well documented although its role has been differently interpreted as being part of the substrate-binding site or the positively charged residue near the N1-C2 = O locus of the flavin coenzyme. To have a better insight into the role of the guanidinium group in D-amino-acid oxidase we have carried out inactivation studies using phenylglyoxal as an arginine-directed reagent. Loss of catalytic activity followed pseudo-first-order kinetics for the apoprotein whereas the holoenzyme showed a biphasic inactivation pattern. Benzoate had no effect on holoenzyme inactivation by phenylglyoxal and the coenzyme analog 8-mercapto-FAD did not provide any additional protection in comparison to the native coenzyme. Spectroscopic experiments indicated that the modified protein is unable to undergo catalysis owing to the loss of coenzyme-binding ability. Analyses of time-dependent activity loss versus arginine modification or [14C]phenylglyoxal incorporation showed the presence of one arginine essential for catalysis. The protection exerted by the coenzyme is consistent with the involvement of an active-site arginine in the correct binding of FAD to the protein moiety. Comparative analyses of CNBr fragments obtained from apoenzyme, holoenzyme and the 8-mercapto derivative of D-amino-acid oxidase after reaction with phenylglyoxal did not provide unequivocal identification of the essential arginine residue within the primary structure of the enzyme. However, they suggest that it might be localized in the N-terminal portion of the polypeptide chain and point to a role of phenylglyoxal-modifiable arginine in binding to the adenylate/pyrophosphate moiety of the flavin coenzyme.