No abstract

A number of proteins have been rendered functionally oestrogen-dependent by fusion with the hormone-binding domain of the oestrogen receptor. There are, however, several significant disadvantages with such fusion proteins. First, their use in cells in vitro requires phenol red-free medium and laborious stripping of steroid hormones from serum in order to avoid constitutive activation. Secondly, control of oestrogen receptor fusion proteins in vivo is precluded by high endogenous levels of circulating oestrogens. Thirdly, the hormone-binding domain of the oestrogen receptor functions as a hormone-dependent transcriptional activation domain making interpretation of fusions with transcription factors problematical. In order to overcome these drawbacks we have used a transcriptionally inactive mutant of the murine oestrogen receptor which is unable to bind oestrogen yet retains normal affinity for the synthetic ligand, 4-hydroxytamoxifen. When the hormone-binding domain of this mutant oestrogen receptor is fused to the C-terminus of the c-Myc protein, Myc-induced proliferation and apoptosis in fibroblasts becomes dependent on 4-hydroxytamoxifen, but remains refractory to 17 beta-oestradiol.

Bim (Bcl-2-interacting mediator of cell death) is a member of the BH3 domain-only subgroup of Bcl-2 family members, for which three splice variants have been described. Bim is expressed in many healthy cell types, where it is maintained in an inactive conformation through binding to the microtubule-associated dynein motor complex. Upon certain apoptotic stimuli, Bim is released from microtubules and mediates caspasedependent apoptosis through a mechanism that is still unclear. Here, we have identified and characterized novel splice variants of human Bim mRNA. In particular, we show that a newly discovered, small protein isoform, BimAD, is also able to induce apoptosis strongly in several human cell lines. BimAD and the previously characterized isoform BimS are shown to be capable of heterodimerizing in vivo with both death antagonists (Bcl-2 and Bcl-X L ) and death agonists (Bax). Mutants of BimAD that bind to Bax but not to Bcl-2 still promote apoptosis, indicating that Bim can regulate apoptosis through direct activation of the Baxmediated cell death pathway without interaction with antiapoptotic Bcl-2 family members. Furthermore, we have shown that the interaction of the BimS and BimAD isoforms with Bax leads to a conformational change in this protein analogous to that triggered by the BH3-only protein Bid.

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.

SUMMARYFour monoclonal antibodies to glycoprotein D (gD) of herpes simplex virus (HSV) types 1 and 2 neutralized virus in the presence of complement but exhibited diverse activities in its absence. Amino acid substitutions that conferred resistance to neutralization by each antibody were identified by deriving the nucleotide sequence of the gD gene from resistant mutants. Each antibody selected a substitution from different parts of the molecule and mutants resistant to a single antibody always arose from the same mutation. One of the antibodies reacted with a synthetic oligopeptide corresponding to the region of the molecule in which amino acid substitution conferred resistance, but the remaining three antibodies failed to react with predicted oligopeptide targets. These antibodies may therefore react with 'discontinuous' epitopes, a view supported by the observation that two of these three antibodies competed with each other in binding assays despite the fact that substitutions conferring resistance to neutralization arose nearly 100 residues apart in the primary sequence. The four antibodies had very different biological properties. One antibody neutralized infectivity but did not inhibit cell fusion, one antibody inhibited cell fusion but did not neutralize, while a third antibody had both activities. One antibody had neither activity but enhanced the infectivity of HSV-2 in a type-specific manner. The ability of antibodies to inhibit cell fusion by syncytial virus strains correlated with an ability to prevent plaque enlargement by a non-syncytial virus strain, implying a role for gD in the intercellular spread of virus that is independent of the syncytial phenotype. We found no correlation between neutralizing activity and anti-fusion activity suggesting that, while gD is involved in cell fusion, it has at least one other function which is required for infectivity.

KRAS represents an excellent therapeutic target in lung cancer, the most commonly mutated form of which can now be blocked using KRAS-G12C mutant-specific inhibitory trial drugs. Lung adenocarcinoma cells harboring KRAS mutations have been shown previously to be selectively sensitive to inhibition of mitogen-activated protein kinase kinase (MEK) and insulin-like growth factor 1 receptor (IGF1R) signaling. Here, we show that this effect is markedly enhanced by simultaneous inhibition of mammalian target of rapamycin (mTOR) while maintaining selectivity for the KRAS-mutant genotype. Combined mTOR, IGF1R, and MEK inhibition inhibits the principal signaling pathways required for the survival of KRAS-mutant cells and produces marked tumor regression in three different KRAS-driven lung cancer mouse models. Replacing the MEK inhibitor with the mutant-specific KRAS-G12C inhibitor ARS-1620 in these combinations is associated with greater efficacy, specificity, and tolerability. Adding mTOR and IGF1R inhibitors to ARS-1620 greatly improves its effectiveness on KRAS-G12C mutant lung cancer cells in vitro and in mouse models. This provides a rationale for the design of combination treatments to enhance the impact of the KRAS-G12C inhibitors, which are now entering clinical trials.

Using a panel of non-small cell lung cancer (NSCLC) lines, we show here that MAP-ERK kinase (MEK) and RAF inhibitors are selectively toxic for the KRAS -mutant genotype, whereas phosphoinositide 3-kinase (PI3K), AKT, and mTOR inhibitors are not. IGF1 receptor (IGF1R) tyrosine kinase inhibitors also show selectivity for KRAS -mutant lung cancer lines. Combinations of IGF1R and MEK inhibitors resulted in strengthened inhibition of KRAS -mutant lines and also showed improved effectiveness in autochthonous mouse models of Kras -induced NSCLC. PI3K pathway activity is dependent on basal IGF1R activity in KRAS -mutant, but not wild-type, lung cancer cell lines. KRAS is needed for both MEK and PI3K pathway activity in KRAS -mutant, but not wild-type, lung cancer cells, whereas acute activation of KRAS causes stimulation of PI3K dependent upon IGF1R kinase activity. Coordinate direct input of both KRAS and IGF1R is thus required to activate PI3K in KRAS -mutant lung cancer cells. SIGNIFICANCE: Ithas not yet been possible to target RAS proteins directly, so combined targeting of effector pathways acting downstream of RAS, including RAF/MEK and PI3K/AKT, has been the most favored approach to the treatment of RAS -mutant cancers. This work sheds light on the ability of RAS to activate PI3K through direct interaction, indicating that input is also required from a receptor tyrosine kinase, IGF1R in the case of KRAS -mutant lung cancer. This suggests potential novel combination therapeutic strategies for NSCLC. Cancer Discov; 3(5);

Oncogenic mutations in RAS genes are very common in human cancer, resulting in cells with well-characterized selective advantages, but also less well-understood vulnerabilities. We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells, but not by derivatives lacking this oncogene. Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells. Cancer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6. Extending this analysis using a collection of drugs with known targets, we found that cancer cells with mutant KRAS showed selective addiction to proteasome function, as well as synthetic lethality with topoisomerase inhibition. Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells. These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers, which are traditionally seen as being highly refractory to therapy.