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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. 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. 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.

The new Consensus Genomic Staging (CGS) of high-risk multiple myeloma (MM) was recently updated by the International Myeloma Society and International Myeloma Working Group. We performed a retrospective study to validate the revised HRMM criteria in relapsed and refractory MM patients treated with BCMA CAR-T. A total of 158 patients were included, of whom 75 (47%) met criteria for HRMM. The median follow-up was 24.8 months (95% CI 22.5–30.3). On multivariable analysis, patients with HRMM had significantly shorter PFS (HR 1.89, p  = 0.003) and OS (HR 3.06, p  < 0.001) compared to standard-risk MM (SRMM) following BCMA CAR-T. LDH > 210 U/L and EMD were also identified as independent adverse risk factors. Based on survival analyses, we constructed a reclassified staging system incorporating LDH and EMD into the CGS-HRMM criteria. Patients were divided into four discrete risk groups: low risk (SRMM with normal LDH and no EMD; n  = 42, 26%), intermediate-low risk (HRMM with normal LDH and no EMD; n  = 31, 20%), intermediate-high risk (SRMM with high LDH, EMD, or both; n  = 41, 26%), and high risk (HRMM with high LDH, EMD, or both; n  = 44, 28%). Patients with low, intermediate-low, intermediate-high, and high-risk disease had a median PFS of 35.0, 19.6, 10.4, and 6.6 months, respectively ( p  < 0.0001), and an estimated 24-month OS of 90%, 69%, 66%, and 26%, respectively ( p  < 0.0001). In summary, we validated the new CGS-HRMM criteria in relapsed and refractory MM patients treated with BCMA CAR-T. We also propose a reclassified staging system to further improve risk stratification in MM patients treated with BCMA CAR-T.

Background Th17 cells are CD4+ cells that produce interleukin 17 (IL-17) and are potent inducers of tissue inflammation and autoimmunity. We studied the levels of this T cell subset in peripheral blood of patients treated with the anti-CTLA4 antibody tremelimumab since its major dose limiting toxicities are inflammatory and autoimmune in nature. Methods Peripheral blood mononuclear cells (PBMC) were collected before and after receiving tremelimumab within two clinical trials, one with tremelimumab alone (21 patients) and another together with autologous dendritic cells (DC) pulsed with the melanoma epitope MART-1 26–35 (6 patients). Cytokines were quantified directly in plasma from patients and after in vitro stimulation of PBMC. We also quantified IL-17 cytokine-producing cells by intracellular cytokine staining (ICS). Results There were no significant changes in 13 assayed cytokines, including IL-17, when analyzing plasma samples obtained from patients before and after administration of tremelimumab. However, when PBMC were activated in vitro , IL-17 cytokine in cell culture supernatant and Th17 cells, detected as IL-17-producing CD4 cells by ICS, significantly increased in post-dosing samples. There were no differences in the levels of Th17 cells between patients with or without an objective tumor response, but samples from patients with inflammatory and autoimmune toxicities during the first cycle of therapy had a significant increase in Th17 cells. Conclusion The anti-CTLA4 blocking antibody tremelimumab increases Th17 cells in peripheral blood of patients with metastatic melanoma. The relation between increases in Th17 cells and severe autoimmune toxicity after CTLA4 blockade may provide insights into the pathogenesis of anti-CTLA4-induced toxicities. Trial Registration Clinical trial registration numbers : NCT0090896 and NCT00471887

Background A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines. Materials and methods The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot. Results Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRAS Q61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAF V600E mutants, including highly synergistic effects in two BRAF V600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAF V600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways. Conclusions The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAF V600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

Background TAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2. Methods The growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733. Results Fourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAF V600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRAS Q61L mutant and GNAQ mutant cell lines than in BRAF V600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays. Conclusions The MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3′ dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae. The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.

Drug-resistance mechanism in melanoma Clinical trials in melanoma patients carrying B-RAF gene mutations have shown promising results with the B-RAF kinase inhibitor PLX4032, but many patients go on to become resistant. Two papers now uncover possible mechanisms for this resistance. Nazarian et al . report that melanomas can acquire resistance due to mutations of N-RAS or increased expression of PDGFRβ , and Johannessen et al . report resistance due to upregulation of MAP3K8/COT. Each of these mechanisms seems to apply to some patients in the recent PLX4032 trial, yet surprisingly, no secondary B-RAF mutations were observed. Recent data from early clinical trials in melanoma patients carrying mutations in the B-RAF gene have shown promising results with the B-RAF kinase inhibitor PLX4032; however, many patients eventually develop resistance to this treatment. Two papers now uncover possible mechanisms of resistance to PLX4032. One paper shows that upregulation of MAP3K8 (which encodes COT) can confer resistance of melanoma cells to B-RAF inhibitors, whereas another paper found that melanomas can acquire resistance due to mutations of N-RAS or increased expression of PDGFRβ. Each of these resistance mechanisms seems to apply to at least some patients on recent PLX4032 trial, whereas, surprisingly, so far no secondary B-RAF mutations have been observed. Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ∼7% of human malignancies and ∼60% of melanomas 1 . Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses 2 . Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E) , MAPK reactivation, and activation of alternative survival pathways 3 , 4 , 5 . Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS ) mutations but not through secondary mutations in B-RAF(V600E) . We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E) -positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

Blocking oncogenic signaling induced by the BRAFV600E 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 BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 muM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 muM, and three were moderately sensitive with IC50 values between 1 and 10 muM. 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, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.

Histones have long been known for their functions in packaging the eukaryotic genome and in regulating DNA-based processes. These functions are dependent on physical and chemical properties of histones constructing the nucleosome, the basic structural unit for packaging DNA. The packaging of DNA into chromatin limits DNA accessibility for processes such as transcription and DNA replication, putting the nucleosome in a position to govern and fine-tune these processes. The influence on DNA metabolic processes can be mediated through alterations in nucleosome composition and positioning as well as covalent modification of histones. Variations in nucleosome structure and stability achieved by the use of non-canonical histone variants can lead to changes in DNA wrapping and accessibility. Similarly, covalent modifications of histone N-terminal or core domains can lead to structural changes for the nucleosome and the chromatin fiber. Covalent modification of histone tails also contributes to recruitment and anchoring of protein complexes required for transcription, DNA repair and replication. Changes in histone modifications and mutations in the enzymes adding and removing them are implicated in a wide-range of pathologies including cancer (discussed in chapters II, III and IV). Histone modifications are dynamically regulated, which makes them suitable for integration of environmental and cellular cues with gene expression. While histone modifications locally participate in orchestrating transcription, global changes in histone modifications can have profound effects on cellular physiology and metabolism. In fact, alterations in cellular levels of histone modifications are observed in diseases such as cancer and can be responsive to changes in physiologic state of the cell like intracellular pH. In addition to the well-recognized structural elements of histones, a less well-known feature of these ancient proteins is their potential metal binding capabilities. Since metals such as copper and iron are required for a variety of important functions, a potential ability of histones to affect cellular metal homeostasis would greatly increase their influence on cellular functions. Indeed we have recently discovered the function of one such metal binding site at the nucleosome core within the H3 dimerization interface in regulating copper homeostasis. Investigating the role of this region of the nucleosome in metal biology revealed an unprecedented molecular function of the nucleosome as an oxidoreductase enzyme, capable of catalyzing the reduction of Cu2+ to its biousable form Cu 1+. This remarkable enzymatic activity significantly affects copper- dependent activities including mitochondrial respiration and Sod1 function, with a profound impact on the molecular biology of eukaryotes and potentially their evolutionary origin. The importance of copper for various physiological and pathological states in humans further underscores the broad implications of copper reductase activity of the nucleosome. These discoveries mark a new frontier into chromatin biology and a function of histones which may have deemed these proteins suitable participants in the emergence of eukaryotes on Earth (discussed in chapters V and VI).

A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines. The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot. Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRAS.sup.Q61K .sup.mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAF.sup.V600E .sup.mutants, including highly synergistic effects in two BRAF.sup.V600E .sup.mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAF.sup.V600E .sup.mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways. The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAF.sup.V600E .sup.mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.