Dependence of tumor cell lines and patient-derived tumors on the NAD salvage pathway renders them sensitive to NAMPT inhibition with GNE-618.

Nicotinamide adenine dinucleotide (NAD) is a critical metabolite that is required for a range of cellular reactions. A key enzyme in the NAD salvage pathway is nicotinamide phosphoribosyl transferase (NAMPT), and here, we describe GNE-618, an NAMPT inhibitor that depletes NAD and induces cell death in vitro and in vivo. While cells proficient for nicotinic acid phosphoribosyl transferase (NAPRT1) can be protected from NAMPT inhibition as they convert nicotinic acid (NA) to NAD independent of the salvage pathway, this protection only occurs if NA is added before NAD depletion. We also demonstrate that tumor cells are unable to generate NAD by de novo synthesis as they lack expression of key enzymes in this pathway, thus providing a mechanistic rationale for the reliance of tumor cells on the NAD salvage pathway. Identifying tumors that are sensitive to NAMPT inhibition is one potential way to enhance the therapeutic effectiveness of an NAMPT inhibitor, and here, we show that NAMPT, but not NAPRT1, mRNA and protein levels inversely correlate with sensitivity to GNE-618 across a panel of 53 non-small cell lung carcinoma cell lines. Finally, we demonstrate that GNE-618 reduced tumor growth in a patient-derived model, which is thought to more closely represent heterogeneous primary patient tumors. Thus, we show that dependence of tumor cells on the NAD salvage pathway renders them sensitive to GNE-618 in vitro and in vivo, and our data support further evaluation of the use of NAMPT mRNA and protein levels as predictors of overall sensitivity.

Live-cell microscopy reveals small molecule inhibitor effects on MAPK pathway dynamics.

Oncogenic mutations in the mitogen activated protein kinase (MAPK) pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2). We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells.