A proteomic study of myeloproliferative neoplasms using reverse-phase protein arrays.

Myeloproliferative neoplasms polycythemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis constitute a group of haematological diseases. The comprehensive assessment of signaling pathway activation in blood cells may aid the understanding of MPN pathophysiology. Thus, levels of post-translational protein modifications and total protein expression were determined in MPN patients and control leukocytes by using reverse-phase protein arrays (RPPA). Compared to control samples, p-SRC, p-CTNNB1, c-MYC, MCL-1, p-MDM2, BAX and CCNB1 showed higher expression in PV samples than controls. P-JAK2/JAK2 and pro-apoptotic BIM showed differential expression between JAK2V617F-positive and -negative ET patients. Apoptosis, cancer and PI3K/AKT pathways proteins showed differential expression among the studied groups. For most of the proteins analyzed using Western-Blot and RPPA, RPPA showed higher sensitivity to detect subtle differences. Taken together, our data indicate deregulated protein expression in MPN patients compared to controls. Thus, RPPA may be a useful method for broad proteome analysis in MPN patients´ leukocytes.

Continuous low plasma concentrations of everolimus provides equivalent efficacy to oral daily dosing in mouse xenograft models of human cancer.

PURPOSE: Everolimus is a drug used successfully in a number of different oncology indications, but significant on-target toxicities exist. We explored the possibility of improving the therapeutic index (TI) by studying alternative means of administering the drug based upon low continuous dosing. METHODS: All studies were performed using naïve nude mice or nude mice bearing s.c. human renal 786-O tumours or human breast MDA-MB-468 tumours. Everolimus was administered via a standard emulsion, either i.v., p.o., i.p., s.c., or via s.c. osmotic mini-pumps (MP) or via poly-lactic-co-glycolic (PLGA)-microparticles (PLGA-µP) prepared from everolimus powder injected s.c. Total-drug levels in blood, plasma or tissues were quantified ex vivo by LC-MS/MS. Efficacy studies were performed over 2-3 weeks and toxicity assessed by changes in body weight, glucose and white blood cell count. Effects on tumour activity biomarkers were quantified using reverse-phase protein array. RESULTS: Everolimus administration s.c. in an emulsion decreased the absorption rate but increased the C max and bio-availability of everolimus compared to standard approaches of administration p.o. or i.p. Everolimus administration s.c. via MP or PLGA-µP reduced the C max and provided continuous low concentrations of everolimus in the plasma, which inhibited tumour pS6/S6 to a similar degree to oral administration. Toxicities such as changes in body weight or white blood cell count were unaffected. Provided the everolimus concentration was above the free unbound IC50 for proliferation of the tumour cell line, efficacy could be achieved equivalent to that provided by standard oral administration. However, an overall improvement in the TI could not be demonstrated. CONCLUSIONS: Continuous low plasma concentrations of everolimus can provide strong efficacy in preclinical models, which if translatable to the clinic may reduce on-target toxicities and so increase the TI.

Maximizing the Efficacy of MAPK-Targeted Treatment in PTENLOF/BRAFMUT Melanoma through PI3K and IGF1R Inhibition.

The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of BRAF-mutant (BRAF(MUT)) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant PTEN loss of function (PTEN(LOF)) occurs in approximately 40% of BRAF(MUT) melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of PTEN(LOF)/BRAF(MUT) melanoma. Large-scale compound sensitivity profiling revealed that PTEN(LOF) melanoma cell lines were sensitive to PI3Kß inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kß inhibitor identified an adaptive response involving the IGF1R-PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kß, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in PTEN(LOF)/BRAF(MUT) melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kß + IGF1R, and MAPK pathway inhibitors in PTEN(LOF)/BRAF(MUT) melanoma patients to achieve maximal response.