SUMMARY PD-1 immune checkpoint blockade provides significant clinical benefits for melanoma patients. We analyzed the somatic mutanomes and transcriptomes of pretreatment melanoma biopsies to identify factors that may influence innate sensitivity or resistance to anti-PD-1 therapy. We find that, while overall high mutational loads associate with improved survival both in responding and non-responding patients, responding tumors are specifically enriched for mutations in the DNA repair gene BRCA2. Innately resistant tumors display a transcriptional signature (referred to as the IPRES or Innate anti-PD-1 Resistance) indicating concurrent upexpression of genes involved in the regulation of mesenchymal transition, cell adhesion, ECM remodeling, angiogenesis and wound-healing. Notably, MAPK-targeted therapy (MAPKi) induces similar signatures in melanoma, suggesting that a non-genomic form of MAPKi resistance mediates cross-resistance to anti-PD-1 therapy. Validation of the IPRES in other independent tumor cohorts defines a transcriptomic subset across distinct types of advanced cancer. These findings suggest that attenuating the biological processes that underlie IPRES may improve anti-PD1 response in melanoma and other cancer types.

The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am. Soc. Mass Spectrom. 2009, 20, 593-596) to enhance multiple charging of native proteins and noncovalent protein complexes in electrospray ionization (ESI) mass spectra. Additional new reagents have been found to "supercharge" proteins from nondenaturing solutions; several of these reagents are shown to be more effective than m-NBA for increasing positive charging. Using the myoglobin protein-protoporphyrin IX (heme) complex, the following reagents were shown to increase ESI charging : benzyl alcohol, m-nitroacetophenone, m-nitrobenzonitrile, o-NBA, m-NBA, p-NBA, m-nitrophenyl ethanol, sulfolane (tetramethylene sulfone), and m-(trifluoromethyl)-benzyl alcohol. Based on average charge state, sulfolane displayed a greater charge increase (61%) than m-NBA (21%) for myoglobin in aqueous solutions. The reagents that promote higher ESI charging appear to have low solution-phase basicities and relatively low gas-phase basicities, and are less volatile than water. Another feature of mass spectra from some of the active reagents is that adducts are present on higher charge states, suggesting that a mechanism by which proteins acquire additional charge involves direct interaction with the reagent, in addition to other factors such as surface tension and protein denaturation. (J Am Soc Mass Spectrom 2010, 21, 127-131) © 2010 American Society for Mass Spectrometry E lectrospray ionization is distinguished among other desorption/ionization sources for mass spectrometry by its ready generation of multiply charged molecules, the value of which was recognized early by Fenn's group: "This feature is very attractive in that it extends the effective mass range of any analyzer by a factor equal to the number of charges per ion. Moreover, because the ions have lower m/z values, they are generally easier to detect and weigh than are singly charged ions of the same mass [1]."After the utility of ESI-MS for protein analysis was demonstrated, multiple charging was rapidly extended to tandem mass spectrometry of large peptides and proteins [2,3]. Dissociation efficiency of large molecules is enhanced with increasing charge, allowing sequenceinformative product ions to be measured. Early ESI-MS studies investigated the potential of different solvents to increase charging [4]. However, useful enhancement of ESI multiple charging was not described clearly until Iavarone and Williams reported "supercharging" promoted by agents such as m-nitrobenzyl alcohol (m-NBA) [5,6]. For protein analyses, their work largely investigated solutions considered to be denaturing.Recently, we demonstrated that multiple charging of noncovalent protein complexes could be increased in ESI-MS when spectra are obtained from nondenaturing protein solutions containing up to 1% (vol/vol) m-NBA [7]. Increases in charge ranged from 8% for the 690 kDa 20S proteasome complex to 48% additional charge for zinc-bound 29 kDa carbonic anhydrase protein. These protein structures were cons...

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Treatment of advanced V600BRAF mutant melanoma using a BRAF inhibitor (BRAFi) or its combination with a MEKi typically elicits partial responses. We compared the transcriptomes of patient-derived tumors regressing on MAPKi therapy against MAPKi-induced temporal transcriptomic states in human melanoma cell lines or murine melanoma in immune-competent mice. Despite heterogeneous dynamics of clinical tumor regression, residual tumors displayed highly recurrent transcriptomic alterations and enriched processes, which were also observed in MAPKi-selected cell lines (implying tumor cell-intrinsic reprogramming) or in bulk mouse tumors (and the CD45-negative or –positive fractions,, implying tumor cell-intrinsic or stromal/immune alterations, respectively). Tumor cell-intrinsic reprogramming attenuated MAPK-dependency, while enhancing mesenchymal, angiogenic and IFN-inflammatory features and growth/survival dependence on multi-RTKs and PD-L2. In the immune compartment, PD-L2 upregulation in CD11c+ immunocytes drove the loss of T-cell inflammation and promoted BRAFi resistance. Thus, residual melanoma early on MAPKi therapy already displays potentially exploitable adaptive transcriptomic, epigenomic, immune-regulomic alterations.

Increased multiple charging of native proteins and noncovalent protein complexes is observed in electrospray ionization (ESI) mass spectra obtained from nondenaturing protein solutions containing up to 1% (v/v) m-nitrobenzyl alcohol (m-NBA). The increases in charge ranged from 8% for the 690 kDa α 7 β 7 β 7 α 7 20S proteasome complex to 48% additional charge for the zinc-bound 29 kDa carbonic anhydrase-II protein. No dissociation of the noncovalently bound ligands/subunits was observed upon the addition of m-NBA. It is not clear if the enhanced charging is related to altered surface tension as proposed in the "supercharging" model of Iavarone and Williams (Iavarone, A. T.; Williams, E. R. J. Am. Chem. Soc. 2003, 125, 2319-2327. However, more highly charged noncovalent protein complexes have utility in relaxing slightly the mass-to-charge (m/z) requirements of the mass spectrometer for detection and will be effective for enhancing the efficiency for tandem mass spectrometry studies of protein complexes.