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PI3Kα is highly mutated in cancer resulting in hyperactivation of lipid kinase activity and downstream AKT signaling. H1047 is the most common site of oncogenic mutation and occurs in ~14% of all breast cancers. Initial therapeutic benefit of targeting PI3Kα was established with alpelisib, an alpha-selective PI3K inhibitor that is equipotent against wild-type and mutant forms. However, wild-type PI3Kα inhibition results in frequent dose-limiting toxicities including hyperglycemia, restricting the full potential of this drug. Selective targeting of H1047X-mutant PI3Kα is expected to both improve anti-tumor activity and reduce toxicity. STX-478 is an allosteric, CNS-penetrant, selective PI3Kα H1047X inhibitor, having excellent drug-like properties and exceptional kinome selectivity. STX-478 demonstrated minimal inhibition of CYP enzymes in vitro, supporting the potential for combinations with a wide range of therapeutics in breast cancer and a variety of other tumor types. STX-478 selectivity extended to the inhibition of other activating kinase domain mutations in biochemical assays. In a diverse panel of PI3Kα H1047X mutant cell lines, STX-478 selectively reduced the cellular levels of pAKT (S473) with a strong correlation between pAKT inhibition and cell viability (R = 0.8). In a high-throughput viability screen of 467 cancer cell lines, the presence of PIK3CA H1047X and other kinase domain mutations were the single strongest predictor of STX-478 sensitivity with potency superior to alpelisib. STX-478 also selectively inhibited the proliferation of cell lines with PI3Kα helical domain mutations, potentially due to the selective dependency of these cells on mutant PI3Kα. When combined with fulvestrant, lapatinib, or abemaciclib, STX-478 demonstrated synergistic anti-proliferative activity in cell lines with relevant ER/HER2 status. Unlike alpelisib, STX-478 did not impair glucose metabolism or cause insulin resistance at efficacious doses. In the T47D (PI3Kα H1047R) breast cancer model, STX-478 (100 mg/kg) monotherapy caused tumor regression whereas alpelisib caused only stasis. STX-478 combination with fulvestrant was well-tolerated, with more consistent and deeper tumor regression. Similar results were observed in a PI3Kα H1047R mutant ER+/HER2- PDX model, where fulvestrant monotherapy showed minimal activity, while combination with STX-478 yielded tumor regressions. In an ER+/HER2+ PDX model (PI3Kα H1047R/R108H), palbociclib and STX-478 (100 mg/kg) monotherapy resulted in similar efficacy while the combination was well tolerated and yielded tumor regression. Together these data indicate robust STX-478 monotherapy activity that was well tolerated and improved when dosed in combination with fulvestrant or CDK4/6 inhibitors. Finally, we investigated the effect of STX-478 treatment in an ER+ PDX model carrying a helical domain mutation. STX-478 treatment resulted in tumor growth inhibition at doses that did not result in metabolic dysfunction, suggesting that STX-478 may also be efficacious in treating PIK3CA mutant tumors with helical domain mutations. In summary, STX-478 efficacy was superior to alpelisib at a dose level that exceeds the clinically relevant exposure in mice without causing metabolic dysfunction. STX- 478 has a predicted low human dose, CNS exposure, low risk of DDI, and a predicted long half-life with minimal variation in peak-to-trough plasma concentrations which further supports a favorable therapeutic index. STX-478 has the potential to provide a best-in-class profile to improve outcomes in patients harboring tumors with prevalent PI3Kα H1047X mutations as well as other kinase and helical domain mutant tumors. The significant CNS exposure of STX-478 is expected to enable this treatment for patients with brain tumors and brain metastases not afforded by existing options. STX-478 is currently in IND enabling studies and is expected to enter human clinical trials in 2023. Citation Format: Leonard Buckbinder, David J. St. Jean, Brendon Ladd, Trang Tieu, Philip Jonsson, Jacob Alltucker, Samantha Manimala, Weixue Wang, Angel Guzman-Perez, Darrin D. Stuart, Gregory Dowdell. STX-478, a mutant-selective PI3Kα H1047X inhibitor clinical candidate with a best-in-class profile: Pharmacology and therapeutic activity as monotherapy and in combination in breast cancer xenograft models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-04.

Supplementary Figure S1 shows genetic dependency profiles for BRAF, MAPK1 (ERK2), and MAPK3 (ERK1) by BRAFV600 mutation status from three independent screens: DRIVE, ACHILLES, and AVANA.

The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF–MEK–ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAFV600-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAFV600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation–induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation.Implications:This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.

Inhibitors targeting BRAF and its downstream kinase MEK produce robust response in patients with advanced BRAFV600-mutant melanoma. However, the duration and depth of response vary significantly between patients; therefore, predicting response a priori remains a significant challenge. Here, we utilized the Novartis collection of patient-derived xenografts to characterize transcriptional alterations elicited by BRAF and MEK inhibitors in vivo, in an effort to identify mechanisms governing differential response to MAPK inhibition. We show that the expression of an MITF-high, “epithelial-like” transcriptional program is associated with reduced sensitivity and adaptive response to BRAF and MEK inhibitor treatment. On the other hand, xenograft models that express an MAPK-driven “mesenchymal-like” transcriptional program are preferentially sensitive to MAPK inhibition. These gene-expression programs are somewhat similar to the MITF-high and -low phenotypes described in cancer cell lines, but demonstrate an inverse relationship with drug response. This suggests a discrepancy between in vitro and in vivo experimental systems that warrants future investigations. Finally, BRAFV600-mutant melanoma relies on either MAPK or alternative pathways for survival under BRAF and MEK inhibition in vivo, which in turn predicts their response to further pathway suppression using a combination of BRAF, MEK, and ERK inhibitors. Our findings highlight the intertumor heterogeneity in BRAFV600-mutant melanoma, and the need for precision medicine strategies to target this aggressive cancer.

BRAFV600 mutant melanoma PDXs exhibit varied degrees of MAPK pathway suppression upon BRAF and MEK inhibitor treatment

List of genes from the DEG clustering analysis.

All supplementary figure and table titles and legends.

Supplementary Table 4: List of cell lines evaluated for sensitivity to SHP099 (Tab 1) or an RTK-inhibitor (Tab 2) in 2D or 3D. Included is the lineage and the KRAS mutation status. Where data are blank, no viable data was available, or the cell line did not grow appropriately as a tumor spheroid.

In vivo efficacy of SHP099 (100 mg/kg, daily) in the KYSE-520 esophageal cancer cell line model. Data are plotted as the treatment mean {plus minus} s.e.m (n=7) ( (B-I) In vivo efficacy data for cell line models represented in Fig. 3E. SHP099 and trametinib were orally administered at the doses, schedules and for the duration indicated. Data are plotted as the treatment mean {plus minus} s.e.m.

Figure S1. Cellular phenotypes observed in response to ERK2 overexpression in A-375 cells. Figure S2. No paracrine effect was produced by cells dying due to knockdown of the essential gene PSMA3. Figure S3. A-375 xenograft tumors with ERK2 overexpression displayed necrosis and/or fibrosis. Figure S4. Paradoxical activation triggered by low doses of BRAFi in GR-M augmented the growth suppression caused by ERK2 overexpression. Figure S5. Graphical representations of vector constructs used in this study. Table S1. MSigDB Hallmark pathways that were significantly enriched in genes upregulated after 20 h treatment with doxycycline in A-375 ERK2 cells. Table S2. Transcription factor targets (from MSigDB C3 TFT) for known ERK targets were significantly enriched in genes upregulated after 20 h treatment with doxycycline in A-375 ERK2 cells.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues.Implications:BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

Supplementary Material and Methods. File contains the following: Transcriptome sequencing and analysis, Soft agar assay, 2D and 3D Cell proliferation screen and compound characterization information.

Supplementary Figure 3. (A) Shown are western blots using antibodies directed against T202/Y204 phosphorylated ERK1/2 following incubation with a range of concentrations of LXH254 over a 4 - 72 hour period. Which individual RAF protein is not expressed in each HCT 116 variant is indicated above the relevant panels. (B) Shown are western blots using Abs directed against the indicated proteins in either whole cell lysates (WCL) or material immuno-precipitated from cell lysates using Abs directed against ARAF (ARAF IP) or BRAF (BRAF IP). Lysates were generated from either parental MIA PaCa-2 cells or a MIA PaCa-2 variant lacking expression of CRAF. (C) Recombinant MEK1 can be phosphorylated by immuno-precipitates from both LXH254 treated and untreated MIA PaCa-2 and MEL-JUSO cells using an anti-body directed against ARAF contains MEK1 kinase activity. Proteins detected in either whole cell lysates (WCL) or ARAF-Ab immuno-precipitates (IP:ARAF) are indicated on the right. (D) IP-kinase activity on recombinant MEK1 in immuno-precipitates from HEK293 cells expressing FLAG-tagged ARAF using Abs directed against FLAG requires ATP and occurs on both wild type (WT) and kinase-dead (K97M) MEK1. (E) Shown are western blots using antibodies directed against T202/Y204 phosphorylated ERK1/2 following incubation with a range of concentrations of LXH254 over a 4-72 hour period. The individual RAF protein expressed in each HCT 116 variant is indicated above the relevant panels. (F) Shown are western blots of phosphorylated MEK1/2 (S218/S221) and ERK1/2 (T202/Y204) following a 4 hr. incubation with a range of LXH254 concentrations in the RAS/RAF wild type cell line PC-9.

Antiproliferative effect of RAF709 compared to dabrafenib, trametinib, RAF265 and sorafenib across a tumor cell panel

(A) Average body weight of mice treated with each BRAF inhibitor in the experiment depicted in Figure 1C; (B) Encorafenib plasma exposure in mice following the last dose from the experiment depicted in Figure 1C. To estimate daily exposure on the BID regimen, multiply the AUC x 2. Encorafenib is 98.6% protein bound in mouse plasma; (C) Gastric hyperplasia and hyperkeratosis observed in the forestomach of mice treated with BRAFi. Hematoxylin and eosin stains of forestomach sections from mice following 14 days of treatment with vehicle, encorafenib, dabrafenib, or vemurafenib from the study depicted in Figure 1C.

Legends for supplemental figures and tables

Table S1: Kinase hits from KinomeScan panel. Table S4: Antiproliferative activity of RAF709 in a Ba/F3 cell kinase panel.

Purpose:Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254.Experimental Design:LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF.Results:We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive.Conclusions:LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.

Supplementary Legend

Biochemical and cellular potency of encorafenib, dabrafenib, and vemurafenib in biochemical and cell-based assays. Purified BRAF V600E kinase domain was used in the biochemical experiments, using kinase-dead MEK1 as a substrate and a phosphorylated MEK1 (Ser217/221) alpha-screen readout. A375 cells were incubated with inhibitors for 3 hours to determine the phosphorylated ERK (pERK) half maximal effective concentration (EC50) and 72 hours to determine the proliferation EC50

Proliferation IC50 values for various cell lines and cell line derivatives

Purpose: Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with BRAFi treatment-naïve and pretreated BRAF-mutant melanoma.Experimental Design: The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50–700 mg) or twice-daily (75–150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic BRAF-mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib.Results: Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of BRAF V600E–mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar–plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months [95% confidence interval (CI), 7.4–not reached (NR)]. In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9–3.7).Conclusions: Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma. Clin Cancer Res; 23(18); 5339–48. ©2017 AACR.

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor–derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537–48. ©2018 AACR.

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Purpose: The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response.Experimental Design: Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated.Results: Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAFV600E/K), whereas eight of 17 (47%) tumors were BRAF wild type (BRAFWT). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAFWT, of which 1 carried c-KITL576P and another N-RASQ61R mutation, while only 2 (29%) of the responding tumors were BRAFV600E/K. Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11.Conclusions: Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles. Clin Cancer Res; 18(8); 2184–98. ©2012 AACR.

Figure S1: pMEK and pERK suppression in KRAS mutant and BRAFV600E cells by dabrafenib or RAF709. Figure S2: pMEK and pERK suppression in NRAS mutant and BRAFV600E cells by dabrafenib or RAF709. Figure S3: Cell cycle and apoptotic effects of RAF709 in KRASmut cells. Figure S4: Antiproliferative activity of RAF709 in HPAFII cells.

Inhibitors targeting

Background Advanced-stage neuroblastoma resists conventional treatment; hence, novel therapeutic approaches are required. We evaluated the use of c-myb antisense oligodeoxynucleotides (asODNs) delivered to cells via targeted immunoliposomes to inhibit c-Myb protein expression and neuroblastoma cell proliferation in vitro. Methods Phosphorothioate asODNs and control sequences were encapsulated in cationic lipid, and the resulting particles were coated with neutral lipids to produce coated cationic liposomes (CCLs). Monoclonal antibodies directed against the disialoganglioside GD(2) were covalently coupled to the CCLs. (3)H-labeled liposomes were used to measure cellular binding, and cellular uptake of asODNs was evaluated by dot-blot analysis. Growth inhibition was quantified by counting trypan blue dye-stained cells. Expression of c-Myb protein was examined by western blot analysis. Results Our methods produced GD(2)-targeted liposomes that stably entrapped 80%-90% of added c-myb asODNs. These liposomes showed concentration-dependent binding to GD(2)-positive neuroblastoma cells that could be blocked by soluble anti-GD(2) monoclonal antibodies. GD(2)-targeted liposomes increased the uptake of asODNs by neuroblastoma cells by a factor of fourfold to 10-fold over that obtained with free asODNs. Neuroblastoma cell proliferation was inhibited to a greater extent by GD(2)-targeted liposomes containing c-myb asODNs than by nontargeted liposomes or free asODNs. GD(2)-targeted liposomes containing c-myb asODNs specifically reduced expression of c-Myb protein by neuroblastoma cells. Enhanced liposome binding and asODN uptake, as well as the antiproliferative effect, were not evident in GD(2)-negative cells. Conclusions Encapsulation of asODNs into immunoliposomes appears to enhance their toxicity toward targeted cells while shielding nontargeted cells from antisense effects and may be efficacious for the delivery of drugs with broad therapeutic applications to tumor cells.

The effects of repeated exposures to UV-A (335 nm) and UV-B (305 nm) radiation on the crystalline lens were studied by treating cultured bovine lenses daily or weekly. The effects of irradiation on lens optical quality were monitored using an automated scanning laser system that records both relative transmittance and focal length across the lens. Relatively low radiant exposures of UV-B were used (0.06, 0.03, 0.01 J/cm2) compared to UV-A (1.44 J/cm2). In total, 38 treated lenses and 32 controls were cultured for times ranging from 400-1000 hours. Results indicate that this range of UV-B exposure may represent the threshold for in vitro UV-B induced opacification. Lenses treated weekly with 0.06 J/cm2 UV-B showed a significant decrease in transmittance compared to controls 69 hours after the first treatment and an increase in focal length variability. The ability of the lens to repair itself, as found in a previous single dose study, was absent after repeated doses. Lenses exposed daily to 0.03 and 0.01 J/cm2 UV-B showed no significant change in transmittance or focal length variability compared to controls. Daily exposure to 1.44 J/cm2 UV-A resulted in no significant change in transmittance or focal length variability compared to controls.

Cold cataracts were induced in ten bovine lenses and then removed by warming. Cataracts first appeared at an average temperature of 11.7 degrees C. The cataracts appeared to be densest at an average temperature of 1.2 degrees C, while warming caused them to disappear completely at an average temperature of 16.4 degrees C. A computer-operated scanning laser system was used to measure the equivalent focal length and changes in relative transmittance before, during, and after the cataract was induced. In general the focal length profile (spherical aberration) that existed before cooling was recaptured on warming. Scatter values indicate that transmittance is not affected by the temporary cold cataract. Thus the optical performance of the bovine lens appears to be identical before and after cold cataracts are induced. We believe that these results indicate that the cataract has a supramolecular origin.

Young rainbow trout were given diets containing graded levels of methionine for 16 wk. Analysis of the weight gain and food efficiency data showed the methionine requirement to be not more than 0.76% of the diet (1.9% of dietary protein). Activities of regulatory enzymes of the transulfuration pathway, methionine adenosyltransferase and cystathionine synthase in trout liver were not altered by changes in methionine intake. Concentrations of free serine in liver and plasma of the trout were high at low levels of methionine intake but fell as dietary methionine increased. This implied decreased flux through cystathionine synthase at low methionine intakes. Large increases in liver and plasma taurine occurred at high methionine intakes, implying enhanced transulfuration activity. Liver ornithine decarboxylase activity was reduced at the lowest level of dietary methionine used but the activity of S-adenosylmethionine decarboxylase was unchanged. Eye lenses of the trout given these diets were examined by a scanning lens monitor. Analysis of focal length variability with this equipment demonstrated that, if abnormality of the lens is to be avoided, a higher concentration of dietary methionine (0.96% or 0.6% methionine + 0.36% cystine) is needed than that required to maximize growth.

Formulations of antisense oligonucleotides (asODNs) against c-myb or c-myc protooncogenes have been prepared by a new technique that sequesters cationic lipid in the interior of a lipid particle. This technique results in high loading efficiency for the asODNs, small particle size and good stability. When targeted against melanoma cells or neuroblastoma cells via anti-GD 2 coupled at the particle surface, increased cell binding to the cells could be demonstrated. Targeted formulations showed greater inhibition of cell proliferation compared to non-targeted formulations or free drug. Inhibition of cell proliferation was demonstrated to be due to down-regulation of c-myb or c-myc protein expression. The formulations have long-circulation times in vivo, and evaluation for in vivo antitumor activity is currently underway.

The effect of UV-B radiation on the crystalline lens was examined by subjecting bovine lenses in culture to varying low exposures at 300 nm. Lens optical quality was monitored on a long-term basis (to 1000 hrs.) with an automated scanning laser system that recorded both change in relative scatter and focal length across each lens. Data were collected for 20 lens positions at each scan. Radiant exposure levels consisted of 0.5, 0.25, 0.125, 0.06 and 0.03 Jcm-2. Twenty irradiated lenses were compared to twelve untreated controls. All of the irradiated lenses showed changes in scatter and focal length relative to the controls. Most (about 75%) of the treated lenses showed significant increases in scatter (200-400%) and focal length (10-20%) at 40 to 60 hours after exposure. A similar time frame for lens damage was noted by visual inspection. Exposure to UV-B at the above doses did not affect culture longevity.

[This corrects the article DOI: 10.1021/ml5002272.].