Targeted therapy against the BRAF/mitogen-activated protein kinase (MAPK) pathway is a promising new therapeutic approach for the treatment of melanoma. Treatment with selective BRAF inhibitors results in a high initial response rate but limited duration of response. To counter this, investigators propose combining this therapy with other targeted agents, addressing the issue of redundancy and signaling through different oncogenic pathways. An alternative approach is combining BRAF/MAPK-targeted agents with immunotherapy. Preliminary evidence suggests that oncogenic BRAF (BRAF V600E ) contributes to immune escape and that blocking its activity via MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens (MDA). Recognition of MDAs is a critical component of the immunologic response to melanoma, and several forms of immunotherapy capitalize on this recognition. Among the various approaches to inhibiting BRAF/MAPK, broad MAPK pathway inhibition may have deleterious effects on T lymphocyte function. Here, we corroborate the role of oncogenic BRAF in immune evasion by melanoma cells through suppression of MDAs. We show that inhibition of the MAPK pathway with MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors or a specific inhibitor of BRAF V600E in melanoma cell lines and tumor digests results in increased levels of MDAs, which is associated with improved recognition by antigen-specific T lymphocytes. However, treatment with MEK inhibitors impairs T lymphocyte function, whereas T-cell function is preserved after treatment with a specific inhibitor of BRAF V600E. These findings suggest that immune evasion of melanomas mediated by oncogenic BRAF may be reversed by targeted BRAF inhibition without compromising T-cell function. These findings have important implications for combined kinase-targeted therapy plus immunotherapy for melanoma. Cancer Res; 70(13); 5213-9. ©2010 AACR.

Her3 (ErbB3) belongs to the epidermal growth factor receptor tyrosine kinases and is well credentialed as an anti-cancer target but is thought to be “undruggable” using ATP-competitive small molecules because it lacks significant kinase activity. Here we report the first selective Her3 ligand, TX1-85-1, that forms a covalent bond with Cys721 located in the ATP-binding site of Her3. We demonstrate that covalent modification of Her3 inhibits Her3 signaling but not proliferation in some Her3 dependent cancer cell lines. Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3 dependent signaling. Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met. These results suggest that small molecules will be capable of perturbing the biological function of Her3 and the approximately 60 other pseudokinases found in human cells.

The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF⅐p54(nrb) complex cooperates with Ku protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF⅐p54(nrb) complex cooperate to align separate DNA molecules.

Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase.