Ramin Nazarian

Abstract Blocking oncogenic signaling induced by the BRAF V600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAF V600E mutation. Seven out of 10 BRAF V600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC 50 values below 1 μM, and three were moderately sensitive with IC 50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAF V600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.

BACKGROUND: The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF(V600) mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway. METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance. CONCLUSIONS/SIGNIFICANCE: Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.

Late endosomes and lysosomes of mammalian cells in interphase tend to concentrate in the perinuclear region that harbors the microtubule-organizing center. We have previously reported abnormal distribution of these organelles - as judged by reduced percentages of cells displaying pronounced perinuclear accumulation - in mutant fibroblasts lacking BLOC-3 (for ;biogenesis of lysosome-related organelles complex 3'). BLOC-3 is a protein complex that contains the products of the genes mutated in Hermansky-Pudlak syndrome types 1 and 4. Here, we developed a method based on image analysis to estimate the extent of organelle clustering in the perinuclear region of cultured cells. Using this method, we corroborated that the perinuclear clustering of late endocytic organelles containing Lamp1 (for ;lysosome-associated membrane protein 1') is reduced in BLOC-3-deficient murine fibroblasts, and found that it is apparently normal in fibroblasts deficient in BLOC-1 or BLOC-2, which are another two protein complexes associated with Hermansky-Pudlak syndrome. Wild-type and mutant fibroblasts were transfected to express human LAMP1 fused at its cytoplasmic tail to green fluorescence protein (GFP). At low expression levels, LAMP1-GFP was targeted correctly to late endocytic organelles in both wild-type and mutant cells. High levels of LAMP1-GFP overexpression elicited aberrant aggregation of late endocytic organelles, a phenomenon that probably involved formation of anti-parallel dimers of LAMP1-GFP as it was not observed in cells expressing comparable levels of a non-dimerizing mutant variant, LAMP1-mGFP. To test whether BLOC-3 plays a role in the movement of late endocytic organelles, time-lapse fluorescence microscopy experiments were performed using live cells expressing low levels of LAMP1-GFP or LAMP1-mGFP. Although active movement of late endocytic organelles was observed in both wild-type and mutant fibroblasts, quantitative analyses revealed a relatively lower frequency of microtubule-dependent movement events, either towards or away from the perinuclear region, within BLOC-3-deficient cells. By contrast, neither the duration nor the speed of these microtubule-dependent events seemed to be affected by the lack of BLOC-3 function. These results suggest that BLOC-3 function is required, directly or indirectly, for optimal attachment of late endocytic organelles to microtubule-dependent motors.