ROLES FOR BRAF KINASE ACTIVATING MUTATIONS IN MELANOMA: MICROENVIRONMENTAL IMMUNOSUPPRESSION

The BRAF oncogene demonstrates a characteristic mutation (V600E) in a significant fraction of cutaneous melanomas, leading to constitutive activation of the MAP kinase pathway. This genetic lesion endows tumor cells with proliferative and survival advantages, and metastatic melanoma patients treated with the BRAF(V600E)-specific inhibitor, Vemurafenib, have shown dramatic clinical responses. Here, I show that BRAF(V600E) induces transcription of the IL-1α and IL-1β genes in both melanocytes and melanoma cell lines and that this upregulation is specifically abrogated by targeted BRAF(V600E) inhibitors. Furthermore, treatment of melanoma tumor-associated fibroblasts (TAFs) with IL-1α/β significantly enhanced the ability of TAFs to suppress the proliferation and function of melanoma antigen-specific cytotoxic T cells. IL-1α/β treatment of TAFs upregulated multiple immunosuppressive factors, including COX-2 and the PD-1 ligands PD-L1 and PD-L2. Specific BRAF(V600E) inhibitors largely abrogated the ability of melanoma cells to confer T cell-suppressive properties on TAFs. These results support a model in which BRAF(V600E) promotes immune suppression in the melanoma tumor environment through an IL-1-mediated mechanism involving resident stromal fibroblasts. Based on these findings, combination therapies involving targeted BRAF inhibition and T cell-based immunotherapies are warranted.

Intracellular chemical messengers and neuronal function: Two examples

Two distinct classes of neurons have been examined in the nervous system of Aplysia. The membrane properties of these neurons are regulated by intracellular signalling molecules in both a short-term and a long-term fashion.^ The role of the phosphatidylinositol cycle in the control of neuronal properties was studied in a class of bursting pacemaker cells, the left upper-quadrant bursting neurons (cells L2, L3, L4, and L6) of the abdominal ganglion of Aplysia. These cells display a regular burst-firing pattern that is controlled by cyclic changes of intracellular Ca$\sp{2+}$ that occur during the bursting rhythm. The characteristic bursting pattern of these neurons occurs within a range of membrane potentials ($-35$ to $-50$ mV) called the pacemaker range. Intracellular pressure injection of inositol 1,4,5-trisphosphate (IP$\sb3$) altered the bursting rhythm of the bursting cells. Injection of IP$\sb3$ induced a brief depolarization that was followed by a long-lasting (2-15 min) hyperpolarization. When cells were voltage-clamped at potentials within the pacemaker range, injection of IP$\sb3$ generally induced a biphasic response that had a total duration of 2-15 min. An initial inward shift in holding current (I$\sb{\rm in}$), which lasted 5-120 sec, was followed by a slow outward shift in holding current (I$\sb{\rm out}$). At membrane potentials more negative than $-40$ mV, I$\sb{\rm in}$ was associated with a small and relatively voltage-independent increase in membrane conductance. I$\sb{\rm in}$ was not blocked by bath application of TTX or Co$\sp{2+}$. Although I$\sb{\rm in}$ was activated by injection of IP$\sb3$, it was not blocked by iontophoretic injection of ethyleneglycol-bis-(beta-aminoethyl ether), N, N$\sp\prime$-tetraacetic acid (EGTA) sufficient to block the Ca$\sp{2+}$-activated inward tail current (I$\sb{\rm B}$).^ Long-term (lasting at least 24 hours) effects of adenylate cyclase activation were examined in a well characterized class of mechanosensory neurons in Aplysia. The injected cells were analyzed 24 hours later by two-electrode voltage-clamp techniques. We found that K$\sp+$ currents of these cells were reduced 24 hours after injection of cAMP. The currents that were reduced by cAMP were very similar to those found to be reduced 24 hours after behavioral sensitization. These results suggest that cAMP is part of the intracellular signal that induces long-term sensitization in Aplysia. (Abstract shortened with permission of author.) ^

Analysis of Lim1 LIM domains in mouse development

Transcriptional regulation is fundamental for the precise development of all organisms. Through tight regulation, necessary genes are activated at proper spatial and temporal patterns, while unnecessary genes are repressed. A large family of regulator proteins that have been demonstrated to be involved in various developmental processes by activation and repression of target genes is the homeodomain family of proteins. To date, the function of many of these homeoproteins has been elucidated in diverse species. However, the molecular mechanism underlying the function of these proteins has not been fully understood. In this study, the molecular mechanism of the function of a LIM-homeoprotein, Lim1, was examined. In addition to the homeodomain, Lim1 contains two LIM domains that are highly conserved among species. This high conservation along with data from in vitro studies on Xenopus Lim1 suggests that the LIM domains might be important for the function of Lim1 as a transcriptional regulator. Here, the functional importance of the LIM domains of Lim1 was determined by using a novel gene-targeting strategy in mouse embryonic stem (ES) cells. A cre-loxP system was used in conjunction with the unique genomic organization of Lim1 to obtain four types of mutant ES cell lines that would allow for the in vivo analysis of the function of both the LIM domains of Lim1 together and also singularly. These four mutant Lim1 alleles either contained base-pair changes at the LIM encoding exons that alters zinc-binding amino acids of the LIM domains or contained only exogenous loxP sequences in the first intron of Lim1, which serves as the control allele. These mutations in the LIM domains would presumably abolish the zinc-finger tertiary structure of the domain and thus render the domain non-functional. Mice carrying mutations at both the LIM domains of Lim1, L1L2, die around E10 without anterior head structures anterior to rhombomere 3, identical in phenotype to the Lim1 null mutants in spite of the presence of mutant Lim1 RNA. This result demonstrates that the integrity of both the LIM domains are essential for the function of Lim1. This is further supported by the phenotype of mice carrying mutation at only the second LIM domain of Lim1, L2. The L2 mice although still carrying one intact Lim1 LIM domain, also die in utero. The L2 mice die at varying times, from around E8 to E10 with anterior defects in addition to other axial defects which have yet to be fully characterized. The results of this study so far demonstrates that the integrity of both LIM domains are required for the function of Lim1. ^