MTOR: Uncovering the link from nutrients to growth

The mechanisms that regulate organismal growth and coordinate it with the availability of nutrients were unknown until a few decades ago. We now know that one pathway—the mechanistic target of rapamycin (mTOR) pathway—is the major nutrient-sensitive regulator of growth in animals and plays a central role in physiology, metabolism, the aging process, and common diseases. This work describes the development of the mTOR field, from its origins in studies into the mechanism of action of the drug rapamycin to our increasingly sophisticated understanding of how nutrients are sensed.

In my PNAS Inaugural Article, I describe the development of the mTOR field, starting with efforts to understand the mechanism of action of the drug rapamycin, which ∼25 y ago led to the discovery of the mTOR protein kinase. I focus on insights that we have contributed and on work that has been particularly influential to me, as well as provide some personal reflections and stories. We now appreciate that, as part of two distinct complexes, mTORC1 and mTORC2, mTOR is the major regulator of growth (mass accumulation) in animals and is the key link between the availability of nutrients in the environment and the control of most anabolic and catabolic processes. Nutrients signal to mTORC1 through the lysosome-associated Rag GTPases and their many regulators and associated cytosolic and lysosomal nutrient sensors. mTOR signaling is deregulated in common diseases, like cancer and epilepsy, and mTORC1 is a well-validated modulator of aging in multiple model organisms. There is significant excitement around using mTORC1 inhibitors to treat cancer and neurological disease and, potentially, to improve healthspan and lifespan.

I decided to use my PNAS Inaugural Article to write about the development of the mTOR field and to provide some personal recollections that highlight work that has been particularly influential to me. I suppose one writes such pieces when one has been around for a while. This appears to be the case, even though I am still surprised when someone refers to me as senior or I am asked by young scientists to talk about my career.

In the fall of 1992, I went to see Sol Snyder about a thesis project. I remember the meeting well, as I would meet with Sol one-on-one very few times during my time in his laboratory. Sol sat in a comfy office chair in the balled-up way that those of us in his laboratory found impossible to mimic, and he was quiet, knowing the power of silence (we assumed it was a trick he learned during his psychiatry training). I was nervous and blurted out that I wanted to talk about potential projects. After a bit, he said, “Well, David, we work on the brain.” That seemed like a great start, as I wanted to do neuroscience, but then more silence followed, and, as I was to learn, that meant the conversation was over. I left unsettled because the brain was obviously a big topic, meaning I was project-less. That conversation though was likely the most important scientific interaction of my career, as Sol was giving me the freedom to do whatever I wanted, which allowed me to develop my own research direction at a relatively young age. I never did end up working on the brain, but I do take some comfort in having originally purified mTOR from brains.

At that time, others in the laboratory were studying the effects of the immunosuppressant FK506 on neurons and using a structurally related molecule, rapamycin, as a negative control. We were fortunate to have rapamycin because, back then, it was not commercially available, and Sol had obtained it from Suren N. Sehgal at Wyeth-Ayerst Sehgal—widely considered the father of rapamycin and its unrelenting champion until his death in 2003—had purified the compound in 1975 from bacteria found in soil collected on Easter Island. Sehgal had very kindly sent us a large amount, but just as importantly, he had also sent a book titled “Rapamycin Bibliography” with a little note wishing us luck. That book became my inspiration. It consisted mostly of abstracts describing the remarkable antifungal, immunosuppressive, and anticancer effects of rapamycin. It was clear that rapamycin inhibited the proliferation of a wide variety of cells ranging from lymphocytes and cancer cells to various species of yeast and preferentially delayed the G1 phase of the cell cycle. I had just finished the first 2 y of medical school and had learned how the immunosuppressant cyclosporin A was revolutionizing organ transplantation. At that point, I still thought I was going to be a practicing physician, so the medical applications of rapamycin were exciting to me and inspired me to determine how rapamycin works.

Targeted Therapy Drugs for Melanoma Skin Cancer

About half of all melanomas have changes (mutations) in the BRAF gene. Melanoma cells with these changes make an altered BRAF protein that helps them grow. Some drugs target this and related proteins, such as the MEK proteins.

If you have melanoma that has spread beyond the skin, a biopsy sample of it will likely be tested to see if the cancer cells have a BRAF mutation. Drugs that target the BRAF protein (BRAF inhibitors) or the MEK proteins (MEK inhibitors) aren’t likely to work on melanomas that have a normal BRAF gene.

Most often, if a person has a BRAF mutation and needs targeted therapy, they will get both a BRAF inhibitor and a MEK inhibitor, as combining these drugs often works better than either one alone.

Vemurafenib (Zelboraf), dabrafenib (Tafinlar), and encorafenib (Braftovi) are drugs that attack the BRAF protein directly.

These drugs can shrink or slow the growth of tumors in some people whose melanoma has spread or can’t be removed completely.

Dabrafenib can also be used (along with the MEK inhibitor trametinib; see below) after surgery in people with stage III melanoma, where it can help lower the risk of the cancer coming back.

These drugs are taken as pills or capsules, once or twice a day.

Common side effects can include skin thickening, rash, itching, sensitivity to the sun, headache, fever, joint pain, fatigue, hair loss, and nausea. Less common but serious side effects can include heart rhythm problems, liver problems, kidney failure, severe allergic reactions, severe skin or eye problems, bleeding, and increased blood sugar levels.

Some people treated with these drugs develop new squamous cell skin cancers. These cancers are usually less serious than melanoma and can be treated by removing them. Still, your doctor will want to check your skin often during treatment and for several months afterward. You should also let your doctor know right away if you notice any new growths or abnormal areas on your skin.

The MEK gene works together with the BRAF gene, so drugs that block MEK proteins can also help treat melanomas with BRAF gene changes. MEK inhibitors include trametinib (Mekinist), cobimetinib (Cotellic), and binimetinib (Mektovi).

These drugs can be used to treat melanoma that has spread or can’t be removed completely.

Trametinib can also be used along with dabrafenib after surgery in people with stage III melanoma, where it can help lower the risk of the cancer coming back.

Again, the most common approach is to combine a MEK inhibitor with a BRAF inhibitor. This seems to shrink tumors for longer periods of time than using either type of drug alone. Some side effects (such as the development of other skin cancers) are actually less common with the combination.

MEK inhibitors are pills taken once or twice a day.

Common side effects can include rash, nausea, diarrhea, swelling, and sensitivity to sunlight. Rare but serious side effects can include heart lung, or liver damage; bleeding or blood clots; vision problems; muscle damage; and skin infections.

HDAC and HDAC Inhibitor: From Cancer to Cardiovascular Diseases

Histone deacetylases (HDACs) are the group of enzymes that remove the acetyl group from lysine residue. To date, 18 mammalian HDACs have been identified and are characterized into four classes: class I HDACs (HDACs 1, 2, 3, and 8), class II HDACs (HDACs 4, 5, 6, 7, 9, and 10), class IV (HDAC 11) and class III (sirtuin family: sirt1-sirt7). Class II HDACs are further divided into two subgroups: class IIa, which has a large C-terminus, and class IIb, which has two deacetylase domains. Class I, II, and IV HDACs need a zinc ion (Zn2+) and share a similar catalytic core for acetyl-lysine hydrolysis, while class III HDACs require a nicotinamide adenine dinucleotide for their enzyme activity.

Even though both class I and class II HDACs have a conserved HDAC domain, they have quite different characteristics. Class I HDACs are present ubiquitously, whereas class II HDACs are expressed in a tissue-specific manner relatively. The class I HDACs are located mainly in the nucleus, whereas class II HDACs undergo shuttling from the nucleus to the cytoplasm after phosphorylation by protein kinase C or by protein kinase D. The class IV HDAC, HDAC11 shares the catalytic domain both of class I and II HDACs. The specific role of class IV HDAC, however, still remains unclear.

Because of the Zn2+-dependent nature of the HDAC domain in class I, II, and IV HDACs, the intrinsic activity of HDACs is suppressed by inhibitors which occupy the catalytic core of the zinc-binding site. The specific motif of HDAC inhibitor, which fits into the tubular pocket where the Zn2+ existed originally, then interferes with the binding of the Zn2+. HDAC inhibitors can be categorized by the structure of their Zn2+-binding group: hydroxamic acids, carboxylic acid, benzamides, and cyclic peptides. For examples, potent non-selective HDAC inhibitors, trichostatin A (TSA) and suberanilohydroxamic acid (SAHA) are in the hydroxamic acid group, while valproate is a member of the carboxylic acid group, MS-275 is widely used benzamide group inhibitor, and Romidepsin is the representative inhibitor having a cyclic peptide structure.

HDACs have an important role both in transcription regulation and in protein modification. As HDACs remove the acetyl moiety from lysine residues at histone tails, which tighten the interaction between the positive-charged histones and the negative-charged DNA. Therefore, histone deacetylation leads to chromatin compaction and inhibits the binding of transcription machinery at the promoter region, which results in repression of mRNA synthesis. Besides the role in transcription-repression, HDACs also function as regulators in posttranslational modification (PTM). HDACs deacetylate non-histone proteins including both transcription factors, such as E2F, p53, c-Myc, and NF-κB, and signal mediators, such as Stat3, Smad7, and β-catenin which regulate cellular homeostasis. Since PTM determines protein activity, stability, subcellular localization, and protein-protein interactions, these diverse protein modifications by PTM modifiers are important for a proteins fate. PTM determines disease prognosis, and drugs that modulate PTM has been investigated as potentially therapeutic for several pathological disease. Imatinib mesylate successfully interrupts Abl kinase activity by blocking the tyrosine kinase domain and thereby induces apoptosis of malignant cells in chronic myeloid leukemia patients. HDACs are one of the important enzymes that regulate the PTM of other proteins, HDAC inhibitors also have therapeutic potential for refractory diseases. On the basis of the studies demonstrated that HDAC inhibition could reverse cardiovascular diseases, in the present review, we will discuss the role of HDAC and HDAC inhibitors as an important therapeutic target in cardiovascular disease beyond the anticancer properties of HDAC inhibitors.

Combining chemotherapy with epidermal growth factor receptor inhibition in advanced non-small cell lung cancer

Treatment of advanced stage lung cancer is changing rapidly. With the new found knowledge on molecular targets such as the epidermal growth factor receptor (EGFR), effective therapy is now available in a selected population with the target mutation. Single-agent epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is a standard first-line therapy for patients with activating-EGFR mutation such as base-pair deletion in exon 19 or point mutation at exon 21. At the same time, this class of drugs may be combined with chemotherapy. Studies on the concurrent combination of chemotherapy and EGFR-TKI confirmed a lack of efficacy. A phase II study on sequential intercalated combination has demonstrated an improvement in progression-free survival (PFS), but this needs to be validated by the ongoing phase III study. The third approach is to combine EGFR-TKI as maintenance therapy after tumour response or stable disease to cytotoxic chemotherapy. Two phase III studies have shown improvement in PFS, but the use of biomarkers for the selection of maintenance therapy remains debatable. Cetuximab is a monoclonal antibody against EGFR and its combination with chemotherapy was shown to improve overall survival in an unselected population. A new biomarker using the H-score will help to select patients for this combination.

Advanced stage non-small cell lung cancer (NSCLC) continues to be one of the most fatal malignancies. Median overall survival (OS) is persistently lower than 12 months despite optimal platinum-based doublet chemotherapy [Schiller et al. 2002; NSCLC-Meta-Analyses-Collaborative-Group, 2008; Azzoli et al. 2009]. Much work was dedicated to improving the treatment outcome by combining novel drugs to standard platinum-based doublets, but most were not successful. An example that showed superior outcome was the addition of bevacizumab, a monoclonal antibody targeting the vascular endothelial growth factor, to standard chemotherapy [Reck et al. 2009; Sandler et al. 2006]. OS was only modestly increased from 10.3 to 12.3 months (p = 0.003) [Sandler et al. 2006]. The other promising approach is the addition of an epidermal growth factor receptor (EGFR) inhibitor to chemotherapy. In this article we explore the various ways of combining different types of EGFR inhibitors, including tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (Mabs), with chemotherapy in advanced NSCLC. The EGFR inhibitors could be given either concurrently, sequentially in an intercalated approach, or selectively as maintenance therapy. Based on existing data, we also discuss future development and application.