RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4/6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G0/G1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F.

Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells.

Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection.

A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The lux gene cluster of Photorhabdus luminescens was introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.

Aberrant methylation of the estrogen receptor and E-cadherin 5' CpG islands increases with malignant progression in human breast cancer.

Loss of expression for both the estrogen receptor-alpha and E-cadherin genes has been linked to disease progression in human ductal breast carcinomas and has been associated with aberrant 5' CpG island methylation. To assess when, during malignant progression, such methylation begins and whether such methylation increases with advancing disease, we have surveyed 111 ductal carcinomas of the breast for aberrant methylation of the estrogen receptor-alpha and E-cadherin 5' CpG islands. Hypermethylation of either CpG island was evident prior to invasion in approximately 30% of ductal carcinoma in situ lesions and increased significantly to nearly 60% in metastatic lesions. Coincident methylation of both CpG islands also increased significantly from approximately 20% in ductal carcinoma in situ to nearly 50% in metastatic lesions. Furthermore, in all cases, the pattern of methylation displayed substantial heterogeneity, reflecting the well-established, heterogeneous loss of expression for these genes in ductal carcinomas of the breast.

Cellular pharmacology of MTA: a correlation of MTA-induced cellular toxicity and in vitro enzyme inhibition with its effect on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

The mechanism of action of an antifolate may be investigated using a variety of experimental methods. These include experiments in a cell culture setting to observe possible protection against drug effects afforded by the end products of metabolic pathways, assessing the activity of purified target enzymes in the presence of the antifolate, and, finally, the measurement of drug effects on intracellular folate and nucleoside triphosphate pools. The current discussion is focused on studies using CCRF-CEM leukemia cells that were designed to compare and contrast mechanisms of action of the antifolates methotrexate, which is primarily a dihydrofolate reductase inhibitor, raltitrexed, a thymidylate synthase inhibitor, LY309887, a glycinamide ribonucleotide formyltransferase inhibitor, and MTA (multitargeted antifolate), which is a novel antifolate antimetabolite. The results of these studies support the hypothesis that MTA affects multiple enzymatic targets and has a distinct mechanism of action from methotrexate, raltitrexed, and LY309887.

Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethy l]-benzoyl]-L-glutamic acid) is a new folate-based antimetabolite currently in broad phase II clinical evaluation. Previous in vitro studies (C. Shih et al, CancerRes 57: 1116-1123, 1997) have suggested that LY231514 could be a multitargeted antifolate (MTA) capable of inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study compared LY231514 with methotrexate, raltitrexed and a glycinamide ribonucleotide formyltransferase inhibitor, LY309887, at 300, 100, 30 and 100 nM, respectively, for their effects on intracellular folate and at 100, 66, 20 and 30 nM respectively, for their effects on nucleoside triphosphate pools in CCRF-CEM cells. Methotrexate induced an accumulation of dihydrofolate species, together with a rapid depletion of ATP, GTP and all of the deoxynucleoside triphosphates. LY309887 caused an accumulation of 10-formyltetrahydrofolate, a rapid loss of ATP, GTP and dATP, but a slower loss in dCTP, dTTP and dGTP. Both LY231514 and raltitrexed had minimal effects on folate pools. In contrast, they caused rapid depletion of dTTP, dCTP and dGTP, but induced an accumulation of dATP at different rates, with raltitrexed doing so about 2.5 times faster. Most of the observed metabolic changes could be understood on the basis of current knowledge of folate and nucleotide metabolism. We concluded that LY231514 was distinct from methotrexate, LY309887 and raltitrexed based on their metabolic effects in CCRF-CEM cells, and that in this cell line the inhibitory effects of LY231514 were exerted primarily against the thymidylate cycle and secondarily against de novo purine biosynthesis.

Application of automatic image segmentation to tibiae and vertebrae from ovariectomized rats.

Automatic contextual segmentation algorithms were developed to objectively identify bone compartments in pQCT images of tibiae, femora, and vertebrae. Principal advantages of this approach over existing techniques such as histomorphometry are as follows: (a) the algorithms can be implemented in a fast, uniform, nonsubjective manner across many images, allowing unbiased comparisons of therapeutic efficacy; (b) much larger volumes in the region of interest can be analyzed to derive true volumetric parameters for trabecular and cortical bone compartments; and (c) pQCT can be used to quantitate bone effects longitudinally in vivo. An automatic contextual segmentation algorithm was used to analyze over 600 scans of proximal tibiae, distal femora, and L-4 vertebrae from studies with ovariectomized rats. Accuracy and precision analyses were performed, and correlation to histomorphometry parameters showed that pQCT trabecular bone density correlates to Tb.N with r = 0.93, while BV/TV correlates to Tb.N with r = 0.95. In other words, pQCT correlates as well to histomorphometry as histomorphometry does to itself. We conclude that the developed automatic segmentation algorithm provides fast, precise, and objective quantitation of bone compartments that are highly correlated with histomorphometry measurements.

Efficacy of insulin lispro in combination with NPH human insulin twice per day in patients with insulin-dependent or non-insulin-dependent diabetes mellitus. Multicenter Insulin Lispro Study Group.

A common treatment regimen for patients with either insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM) is a combination of rapid-acting insulin and intermediate-acting insulin administered twice each day. It is usually recommended that regular human insulin be injected 30 to 45 minutes before a meal. In practice, patients often inject regular human insulin closer to mealtime, causing a higher post-prandial serum glucose level and an increased potential for hypoglycemia in the postabsorptive period. Insulin lispro, a rapid-acting insulin analogue, is best injected just before a meal because of its more rapid absorption and shorter duration of action. In 707 randomized patients, 379 with IDDM and 328 with NIDDM, we studied the effect of twice-daily insulin lispro or regular human insulin in combination with NPH human insulin (isophane insulin) on premeal, 2-hour postprandial, and bedtime glycemic control. Assessments were based on the results of a seven-point blood glucose profile, the insulin dose (by formulation and time of administration), the incidence and frequency of hypoglycemic episodes, and the glycated hemoglobin value. Treatment with insulin lispro resulted in lower postprandial glucose levels and smaller increases in glucose level after the morning and evening meals compared with treatment with regular human insulin. Overall glycemic control, frequency of hypoglycemic events, and total insulin dose were not different between the two groups. Insulin lispro in combination with NPH human insulin in a twice-per-day regimen allows injection closer to mealtime and improves post-prandial glycemic control without increasing the risk of hypoglycemia.

Protein kinase C isozymes differentially regulate promoters containing PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs.

To investigate the regulation of promoters containing classical phorbol ester response sequences (PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs) by protein kinase C (PKC) isozymes, co-transfections were performed in human dermal fibroblasts with a plasmid containing either the human collagenase promoter or the porcine urokinase plasminogen activator (uPA) promoter linked to the chloramphenicol acetyltransferase gene and a plasmid expressing an individual PKC isozyme. Using this experimental design, seven PKC isozymes were analyzed for their ability to trans-activate the collagenase and uPA promoters. Our results demonstrate that only PKC delta, epsilon, and eta trans-activated the collagenase promoter and that binding of Ap-1 family members to the collagenase 12-O-tetradecanoylphorbol-13-acetate response element (TRE) was not responsible for the isozyme-specific trans-activation. In contrast, the uPA promoter was stimulated by all of the PKC isozymes examined (PKC alpha, betaII, gamma, delta, epsilon, zeta, and eta). These results indicate that PKC isozymes differentially regulate promoters containing PEA-3/TRE motifs and suggest that individual isozymes play unique roles within the cell.