A Hunt for Causes of Atherosclerosis

Using the most advanced tools of molecular biology to sift through the sands of the genome for a little gold.

Blood vessels have a hard life. Every time you put on shoes, the vessels in your feet get smashed and smooshed, for hours on end. And do they complain? Generally, not much. They bounce back and make do with the room you give them. All through the body, vessels are subject to the pumping of the heart, and variations in blood volume brought on by our salt balance. They have to move when we do, and deal with it whenever we sit or lie on them. Curiously, it is the veins in our legs and calves, that are least likely to be crushed in daily life, that accumulate valve problems and go varicose. Atherosclerosis is another, much more serious problem in larger vessels, also brought on by age and injury, where injury and inflammation of the lining endothelial cells can lead to thickening, lipid/cholesterol accumulation, necrosis, calcification, and then flow restriction and fragmentation risk. 

Cross-section of a sclerotic blood vessel. LP stands for lipid pool, while the box shows necrotic and calcified bits of tissue.

The best-known risk factors for atherosclerosis are lipid-related, such as lack of liver re-capture of blood lipids, or lack of uptake around the body, keeping cholesterol and other lipid levels high in the blood. But genetic studies have found hundreds of areas of the genome with risk-conferring (or risk-reducing) variants, most of which are not related to lipid management. These genome-wide association studies (or GWAS) look for correlations between genetic markers and disease in large populations. So they pick up a lot of low-impact genetic variations that are difficult to study, due to their large number and low impact, which can often imply peripheral / indirect function. High-impact variations (mutations) tend to not survive in the population very long, but when found tend to be far more directly involved and informative.

A recent paper harnessed a variety of modern tools and methods to extract more from the poor information provided by GWAS. They come up with a fascinating tradeoff / link between atherosclerosis and cerebral cavernous malformation (CCM), which is distinct blood vessel syndrome that can also lead to rupture and death. The authors set up a program of analysis that was prodigious, and only possible with the latest tools. 

The first step was to select a cell line that could model the endothelial cells at issue. Then they loaded these cells with custom expression-reducing RNA regulators against each one of the ~1600 genes found in the neighborhood of the mutations uncovered by the GWAS analyses above, plus 600 control genes. Then they sequenced all the RNA messages from these single cells, each of which had received one of these "knock-down" RNA regulators. This involved a couple hundred thousand cells and billions of sequencing reads- no simple task! The point was to gather comprehensive data on what other genes were being affected by the genetic lesion found in the GWAS population, and then to (algorithmically) assemble them into coherent functional groups and pathways which could both identify which genes were actually being affected by the original mutations, and also connect them to the problems resulting in atherosclerosis.

Not to be outdone, they went on to harness the AlphaFold program to hunt for interactions among the proteins participating in some of the pathways they resolved through this vast pipeline, to confirm that the connections they found make sense.

They came up with about fifty different regulated molecular programs (or pathways), of which thirteen were endothelial cell specific. Things like angiogenesis, wound healing, flow response, cell migration, and osmoregulation came up, and are naturally of great relevance. Five of these latter programs were particularly strongly connected to coronary artery disease risk, and mostly concerned endothelial-specific programs of cell adhesion. Which makes sense, as the lack of strong adhesion contributes to injury and invasion by macrophages and other detritus from the blood, and adhesion among the endothelial cells plays a central role in their ability / desire to recover from injury, adjust to outside circumstances, reshape the vessel they are in, etc.

Genes near GWAS variations and found as regulators of other endothelial-related genes are mapped into a known pathway (a) of molecular signaling. The color code of changed expression refers to the effect that the marked gene had on other genes within the five most heavily disease-linked programs/pathways. The numbers refer to those programs, (8=angiogenesis and osmoregulation, 48=cell adhesion, 35=focal adhesion, related to cell adhesion, 39=basement membrane, related to cell polarity and adhesion, 47=angiogenesis, or growth of blood vessels). At bottom (c) is a layout of 41 regulated genes within the five disease-related programs, and how they are regulated by knockdown of the indicated genes on the X axis. Lastly, in d, some of these target genes have known effects on atherosclerosis or vascular barrier syndromes when mutated. And this appears to generally correlate with the regulatory effects of the highlighted pathway genes.

"Two regulators of this (CCM) pathway, CCM2 and TLNRD1, are each linked to a CAD (coronary artery disease) risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. ... Specifically, we show that knockdown of TLNRD1 or CCM2 mimics the effects of atheroprotective laminar blood flow, and that the poorly characterized gene TLNRD1 is a newly identified regulator in the CCM pathway."

On the other hand, excessive adhesiveness and angiogenesis can be a problem as well, as revealed by the reverse correlation they found with CCM syndrome. The interesting thing was that the gene CCM2 came up as one of strongest regulators of the five core programs associated with atherosclerosis risk mutations. As can be guessed from its name, it can harbor mutations that lead to CCM. CCM is a relatively rare syndrome (at least compared with coronary artery disease) of localized patches of malformed vessels in the brain, which are prone to rupture, which can be lethal. CCM2 is part of a protein complex, with KRIT1 and PDCD10, and part of a known pathway from fluid flow sensing receptors to transcription regulators (TFs) that turn on genes relevant to the endothelial cells. As shown in the diagram above, this pathway is full of genes that came up in this pathway analysis, from the atherosclerosis GWAS mutations. Note that there is a repression effect in the diagram above (a) between the CCM complex and the MAP kinase cascade that sends signals downstream, accounting for the color reversal at this stage of the diagram.

Not only did they find that this known set of three CCM gene are implicated in the atherosclerosis mutation results, but one of the genes they dug up through their pipeline, TLNRD1, turned out to be a fourth, hitherto unknown, member of the CCM complex, shown via the AlphaFold program to dock very neatly with the others. It is loss of function mutations of genes encoding this complex, which inhibits the expression of endothelial cell pro-cell adhesion and pro-angiogenesis sets of genes, that cause CCM, unleashing these angiogenesis genes to do too much. 

The logic of this pathway overall is that proper fluid flow at the cell surface, as expected in well-formed blood vessels, activates the pathway to the CCM complex, which then represses programs of new or corrective angiogenesis and cell adhesion- the tissue is OK as it is. Conversely, when turbulent flow is sensed, the CCM complex is turned down, and its target genes are turned up, activating repair, revision, and angiogenesis pathways that can presumably adjust the vessel shape to reduce turbulence, or simply strengthen it.

Under this model, malformations may occur during brain development when/where turbulent flow occurs, reducing CCM activation, which is abetted by mutations that help the CCM complex to fall apart, resulting (rarely) in run-away angiogenesis. The common variants dealt with in this paper, that decrease risk of cardiovascular disease / atherosclerosis, appear to have similar, but much weaker effects, promoting angiogenesis, including recovery from injury and adhesion between endothelial cells. In this way, they keep the endothelium tighter and more resistant to injury, invasion by macrophages, and all the downstream sequelae that result in atherosclerosis. Thus strong reduction of CCM gene function is dangerous in CCM syndrome, but more modest reductions are protective in atherosclerosis, setting up a sensitive evolutionary tradeoff that we are clearly still on the knife's edge of. I won't get into the nature of the causal mutations themselves, but they are likely to be diffuse and regulatory in the latter case.

Image of the CCM complex, which regulates response to blood flow, and whose mutations are relevant both to CCM and to atherosclerosis. The structures of TLNRD1 and the docking complex are provided by AlphaFold. 

This method is particularly powerful by being unbiased in its downstream gene and pattern finding, because it samples every expressed gene in the cell and automatically creates related pathways from this expression data, given the perturbations (knockdown of expression) of single target genes. It does not depend on using existing curated pathways and literature that would make it difficult to find new components of pathways. (Though in this case the "programs" it found align pretty closely with known pathways.) On the other hand, while these authors claim that this method is widely applicable, it is extremely arduous and costly, as evidenced by the contribution of 27 authors at top-flight institutions, an unusually large number in this field. So, for diseases and GWAS data sets that are highly significant, with plenty of funding, this may be a viable method of deeper analysis. Otherwise, it is beyond the means of a regular lab.

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Dangerous Memories

Some memory formation involves extracellular structures, DNA damage, and immune component activation / inflammation.

The physical nature of memories in the brain is under intensive scrutiny. The leading general theory is that of positive reinforcement, where neurons that are co-activated strengthen their connections, enhancing their ability to co-fire and thus to express the same pattern again in the future. The nature of these connections has been somewhat nebulous, assumed to just be the size and stability of their synaptic touch-points. But it turns out that there is a great deal more going on.

A recent paper started with a fishing expedition, looking at changes in gene expression in neurons at various time points after the mice were subjected to a fear learning regimen. They took this out to much longer time points (up to a month) than had been contemplated previously. At short times, a bunch of well-known signals and growth-oriented gene expression happened. At the longest time points, organization of a structure called the perineural net (PNN) was read out of the gene expression signals. This is a extracellular matrix sheath that appears to stabilize neuronal connections and play a role in long-term memory and learning. 

But the real shocker came at the intermediate time point of about four days. Here, there was overexpression of TLR9, which is an immune system detector of broken / bacterial DNA, and inducer in turn of inflammatory responses. This led the authors down a long rabbit hole of investigating what kind of DNA fragmentation is activating this signal, how common this is, how influential it is for learning, and what the downstream pathways are. Apparently, neuronal excitation, particularly over-excitation that might be experienced under intense fear conditions, isn't just stressful in a semiotic sense, but is highly stressful to the participating neurons. There are signs of mitochondrial over-activity and oxidative stress, which lead to DNA breakage in the nucleus, and even nuclear perforation. It is a shocking situation for cells that need to survive for the lifetime of the animal. Granted, these are not germ cells that prioritize genomic stability above all else, but getting your DNA broken just for the purpose of signaling a stress response that feeds into memory formation? That is weird.

Some neuronal cell bodies after fear learning. The red dye is against a marker of DNA repair proteins, which form tight dots around broken DNA. The blue is a general DNA stain, and the green is against a component of the nuclear envelope, showing here that nuclear envelopes have broken in many of these cells.

The researchers found that there are classic signs of DNA breakage, which are what is turning on the TLR9 protein, such as seeing concentrated double-strand DNA repair complexes. All this stress also turned on proteases called caspases, though not the cell suicide program that these caspases typically initiate. Many of the DNA break and repair complexes were, thanks to nuclear perforation, located diffusely at the centrosome, not in the nucleus. TLR9 turns on an inflammatory response via NFKB / RELA. This is clearly a huge event for these cells, not sending them into suicide, but all the alarms short of that are going off.

The interesting part was when the researchers asked whether, by deleting the TLR9 or related genes in the pathway, they could affect learning. Yes, indeed- the fear memory was dependent on the expression of this gene in neurons, and on this cell stress pathway, which appears to be the precondition of setting up the perineural net structures and overall stabilization. Additionally, the DNA damage still happened, but was not properly recognized and repaired in the absence of TLR9, creating an even more dangerous situation for the affected neurons- of genomic instability amidst unrepaired DNA.

When TRL9 is knocked out, DNA repair is cancelled. At bottom are wild-type cells, and at top are mouse neurons after fear learning that have had the gene TLR9 deleted. The red dye is against DNA repair proteins, as is the blue dye in the right-most frames. The top row is devoid of these repair activities.

This paper and its antecedent literature are making the case that memory formation (at least under these somewhat traumatic conditions- whether this is true for all kinds of memory formation remains to be seen) has commandeered ancient, diverse, and quite dangerous forms of cell stress response. It is no picnic in the park with madeleines. It is an all-hands-on-deck disaster scene that puts the cell into a permanently altered trajectory, and carries a variety of long-term risks, such as cancer formation from all the DNA breakage and end-joining repair, which is not very accurate. They mention in passing that some drugs have been recently developed against TLR9, which are being used to dampen inflammatory activities in the brain. But this new work indicates that such drugs are likely double-edged swords, that could impair both learning and the long-term health of treated neurons and brains.

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Wherever Did the Pandemic Go?

Covid has attenuated. But is that from its own evolution, or from our immune reactions to it?

Looking at recent gatherings such as the political conventions and the Olympics, it is evident that the pandemic is over. A graph from the CDC says that mortality from Covid-19 is now similar to influenza- not great, but not catastrophic either, running at roughly a thousand deaths a week, and this with negligible public precautions.

Overall mortality of Covid-19 in the US.

A fundamental scientific and policy question about this is why: did the virus evolve to a less virulent state, or have we evolved (or engineered) enough immunity to fend off the worst? Even after the intense focus on this virus and all the research that has been done, this is a difficult question to answer. There has been a parade of variants, one supposedly more virulent and dangerous than the last, except that we are less affected and increasingly able to ignore them. The scientific community is evidently divided on this causal question, with no good ways to test these basic hypotheses.

I am personally very much in the viral evolution camp, believing that this virus has on its own evolved to be less virulent, even as it gained in transmissibility and ability to evade our immune systems. Surveillance of the virus shows quite high levels this summer, even while its effects are minor, overall. The logic is that this kind of virus does not gain from people shutting themselves up at home and being miserable, let alone dying. Much better for us to be surreptitiously infected and infectious, and able to go about our business, at work and play. We recall that Covid was markedly more lethal at the very outset of the pandemic, before the first set of variants developed. Other cold-type viruses seem to have followed a similar path, and the many zoonotic infections we have picked up (including this one) come from other organisms which carry these pathogens without much difficulty, doubtless after a long evolutionary standoff.

But the graph above makes a different argument, since the vaccines came online around the spring of 2021, reached about fifty percent of the population in late 2021, which is followed by the dramatic drop in covid mortality in spring of 2022. Some researchers point to the lack of attenuation of other pathogens, like HIV, tuberculosis, and smallpox, to say that the evolutionary argument does not hold water. After a pathogen has replicated and spread, (in the case of Covid, in the first week of infection, roughly), it doesn't care what happens to the host- literally whether it lives or dies. They would say that it was the immunization campaign that saved us, and continued infection leading to herd immunity that has created a population increasingly resistant to Covid mortality.

Testing these hypotheses would require Covid-naive populations, which would be ideally split into two study sets, one with vaccination followed by infection, and the other infected directly. This kind of thing may happen as a natural experiment somewhere, and perhaps the closest we can come is the release of Covid restrictions in China. In late 2022/early 2023, China switched abruptly from a zero-tolerance policy of social contact and infection, to a zero-tolerance policy towards bad publicity and accurate mortality reporting, while relaxing anti-Covid restrictions. The result was a surge in death rates, to levels estimated to be higher than those elsewhere, including in the US. This argues that during the restrictive period, the virus had not significantly attenuated via its natural evolution, though then the subsequent mass infection and inoculation did eventually lead in China, as it has elsewhere, to the lower mortality rates seen around the world. 

So, despite the rapidity of viral evolution, one has to conclude that over the short term, the immune hypothesis appears superior to the viral evolution hypothesis, as an explanation of general attenuation of Covid mortality. (Robert Kennedy may disagree, of course!) The evolution of virulence is closely related to the whole lifecycle of a pathogen, especially the way it spreads, making comparisons with other pathogens hazardous. Respiratory pathogens have the opportunity to spread without damaging the host too much, and that seems, in principle, like an advantageous evolutionary path. So I would still hypothesize that over the long term, Covid will settle into a less virulent form that triggers less immune activation (the most lethal aspect of Covid infection), in favor of high transmission and co-existence with our immune systems. Other viruses seem to have followed a similar path. How it interacts with further naive populations would be dispositive, though there may not be any left at this point.

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Nominee for Most Amazing Protein: RAD51

On the repair and resurrection of DNA, which gets a lot of help from a family of proteins including RAD51, DMC1, and RecA.

Proteins do all sorts of amazing things, from composing pores that can select a single kind of ion- even just a proton- to allow across a membrane, to massive polymerizing enzymes that synthesize other proteins, DNA, and RNA. There is really no end to it. But one of the most amazing, even incredible, things that happens in a cell is the hunt for DNA homology. Even over a genome of billions of base pairs, it is possible for one DNA segment to find the single other DNA segment that matches it. This hunt is executed for several reasons. One is to line up the homologous chromosomes at meiosis, and carry out the genetic cross-overs between them (when they are lined up precisely) that help scramble our genetic lineages for optimal mix-and-matching during reproduction. Another is for DNA repair, which is best done with a good copy for reference, especially when a full double-strand break has happened. Just this week, a fascinating article showed that memories in our brains depend in some weird way on DNA breaks occurring in neurons, some of which then use the homologous repair process, including homology search, to patch things up.

The protein that facilitates this DNA homology search is deeply conserved in evolution. It is called RecA in bacteria, radA and radB in archaea, and the RAD51 family in eukaryotes. Naturally, the eukaryotic family is most closely related to the archaeal versions (RAD51 and DMC1 evolving from radA, and a series of other, and poorly understood family members, from radB). In this post, I will mostly just call them all RAD51, unless I am referring to DMC1 specifically. The name comes from genetic screens for radiation-sensitive mutants in human and other eukaryotes, since RAD51 plays a crucial role in DNA repair, as noted above. RAD51 is not a huge protein, but it is an ATPase. It binds to itself, forming linear filaments with ATP at the junction points between units. It binds to a single strand of DNA, which is going to be what does the hunting. And it binds, in a complicated way, to another double-stranded DNA, which it helps to open briefly to allow its quality as a target to be evaluated. 

This diagram describes the repair of double strand breaks (DSB) in DNA. First the ends are covered with a bunch of proteins that signal far and wide that something terrible has happened- the cell cycle has to stop.. fire engines need to be called. One of these proteins is RPA, which simply binds all over single-stranded DNA and protects it. Then the RAD51 protein comes in, displaces RPA, and begins the homology search process. The second DNA shown, in dark black, doesn't just happen, but is hunted for high and low throughout the nucleus to find the exact homolog of the broken end. When that exact match is found, the repair process can proceed, with continued DNA synthesis through the lesion, and resolution of the newly repaired double strands, either to copy up the homolog version, or exchange versions (GC, for gene conversion). 

This diagram shows how the notorious (when mutated) oncogene BRCA2 (in green) works. It binds RAD51 (in blue) and brings it, chain-gang style, to the breakpoints of DNA damage to speed up and specify repair.

There have been several structural studies by this point that clarify how RAD51 does its thing. ATP is simply required to form filaments on single-stranded DNA. When a match has been found and RAD51 is no longer needed, ATP is cleaved, and RAD51 falls off, back to reserve status. The magic starts with how RAD51 binds the single stranded DNA. One RAD51 binds for every ~3 bases in the DNA, and the it binds the phosphate backbone, so that the bases are nicely exposed in front, and all stretched out, ready to hunt for matching DNA.

A series of RAD51 molecules (in this case, RecA from bacteria) bound sequentially to single-stranded DNA (red). Note the ATP homolog chemicals in yellow, positioned between each protein unit. One can see that the DNA is stretched out a bit and the bases point outwards.

A closeup view of one of the RAD51 units from above, showing how the bases of the DNA (yellow) are splayed out into the medium, ready to find their partners. They are arranged in orientations similar to how they sit in normal (B-form) DNA, further enhancing their ability to find partners.

The second, and more mysterious part of the operation is how RAD51 scans double-stranded DNA throughout the genome. It has binding sites for double-stranded DNA, away from the single-stranded DNA, and then it also has a little finger that splits open the double-stranded DNA, encouraging separation and allowing one strand to face up to the single stranded DNA that is held firmly by the RAD51 polymer. The transient search happens in eight-base increments, with tighter capture of the double-strand DNA happening when nine bases are matched, and committment to recombination or repair happening when a match of fifteen bases is found.  

These structures show an intermediate where a double-stranded DNA (ends in teal and lavender, and separated DNA segments in green and red) has been captured, making a twelve base match with the stable single-stranded DNA (brown). Note how the double-stranded DNA ends are held by outside portions of the RAD51 protein. Closeup on the right shows the dangling, non-paired DNA strand in red, and the newly matched duplex DNA with green-brown colored base interactions.

These structures can only give a hint of what is going on, since the whole process relies so clearly on the brownian motion that allows super-rapid diffusion of the stablized single-strand DNA+RAD51 over the genome, which it scans efficiently in one-dimensional fashion, despite all the chromatin and other proteins parked all over the place. And while the structures provide insight into how the process happens, it remains incredible that this search can happen, on what is clearly a quite reliable basis, day and day out, as our genomes get hit by whatever the environment throws at us.

"Unfortunately, most RAD51 and RAD51 paralog point mutations that have been clinically identified are classified as variants of unknown significance (VUSs). Future studies to reclassify these RAD51 gene family VUSs as pathogenic or benign are desperately needed, as many of these genes are now included on hereditary breast and ovarian cancer screening panels. Reclassification of HR-deficient VUSs would enable these patients to benefit from therapies that specifically target HR deficiency, as do poly(ADP)-ribose polymerase (PARP) inhibitors in BRCA1/2-deficient cells."

Lastly, one paper made the point that clinicians need better understanding of the various mutations that can affect RAD51 itself. Genetic testing now is able to find all of our mutations, but we don't always know what each mutation is capable of doing. Thus deeper studies of RAD51 will have beneficial effects on clinical diagnosis, when particular mutations can be assigned as disease-causing, thus justifying specific therapies that would otherwise not be attempted.

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Getting Cancer Cells to Shoot Themselves

New chemicals that make novel linkages among cellular components can be powerful drugs.

One theme that has become common in molecular biology over the years is the prevalence of proteins whose only job is to bring other proteins together. Many proteins lack any of the usual jazzy functions, like catalytic enzyme, or ion channel, or signaling kinase, but just serve as "conveners", bringing other proteins together. Typically they are regulated in some way, by phosphorylation, expression, or localization, and some of these proteins serve as key "scaffolds" for the activation of some process, like G-protein activation, or cell cycle control, or cell growth. 

Well, the drug industry has caught on, and is starting to think about chemicals that can do similar things, resulting in occasionally powerful results. Conventional drug design has aimed to bind to whatever protein is responsible for some ill, and inhibit it. Such as an oncogene, or an over-active component of the immune system. This has led to many great drugs, but has significant limitations. The chemical has to bind not just anywhere on the target, but at the particular spot (the active site) that is its business end, where its action happens. And it has to bind really well, since binding and inhibiting only half the target proteins in a cell (or the body) will typically only have a modest effect. These requirements are quite stringent and result in many protein targets being deemed difficult to drug, or "undruggable".

A paradigm for a new kind of chemical drug, which links two functions, is the PROTAC class, which combines binding with a target on one end, with another end that binds to the cell's protein destruction machinery, thereby not just inhibiting the target, but destroying it. A new paper describes an even more nuclear option along this line of drug development, linking an oncogene with a second part that activates the cellular suicide machinery. One can imagine that this approach can have far more dramatic effects.

These researchers synthesize and demonstrate a chemical that binds on one end the oncogene BCL6, mutations of which can cause B cell lymphoma. This gene is a transcription repressor, and orchestrates the development of particular immunologic T cells called T follicular helper cells. One of its roles is to prevent the suicide of these cells when an antigen is present, which is when the cells are most needed. If over-expressed in cancer, these cells think they really need to protect the body and proliferate wildly.

The other end of this chemical, called TCIP1, binds to BRD4, which is another transcription regulator, but this one activates the cell suicide genes, instead of turning them off. Both ends of this molecule were based on previously known structures. The innovation was solely in linking them together. I should say parenthetically that BRD4 is itself recognized as an oncogene, as it can promote cell growth and prevent cell suicide in many settings. So it has ambivalent roles, (inviting a lot of vague writing), and it is somewhat curious that these researchers focused on BRD4 as an apoptosis driver.

"TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1–10 nM in 72 h" 

Here EC50 means the effective concentration where the effect is 50% of maximal. This value of 1.3 nano molar is a very low concentration for a drug, meaning it is highly effective. TP53 is another cancer-driving mutation, common in treatment-resistant cancers. The drug has a characteristic and curious dosage behavior, as its effect decreases at higher concentrations. This is because each individual end of the molecule starts to bind and saturate targets independently, reducing the rate of linkage between the two target proteins, and thus the intended effect.

Chemical structure of TCIP1. The left side binds to BRD4, a regulator of cell suicide, while the right side binds to BCL6, an oncogene.

The authors did numerous controls with related chemicals, and tracked genes that were targeted by the novel chemical, all to show that the dramatic effects they were seeing were specifically caused by the linkage of the two chemical functions. Indeed, BCL6 represses its own transcription in the natural course of affairs, and the new drug reverses this behavior as well, inducing more of its own synthesis, which now potentiates the drug's lethal effect. While the authors did not show effectiveness in animals, they did show that TCIP1 is not toxic in mice. Neither did they show that TCIP1 is orally available, but administered it by injection. But even by this mode, it would, if effective, be a very exciting therapy. Not surprisingly, the authors report a long series of biotech industry ties (rooted at Stanford) and indicate that this technology is under license for drug development.

This approach is highly promising, and a significant advance in the field. It should allow increased flexibility in targeting all kinds of proteins that may or not cause disease, but are specific to or over-expressed in disease states, in order to address those diseases. It will allow increased flexibility in targeting apoptosis (cell suicide) pathways through numerous entry points, to have the same ultimate (and highly effective) therapeutic endpoint. It allows drugs to work at low concentrations, not needing to fully occupy or inhibit their targets. Many possible areas of therapy can be envisioned, but one is aging. By targeting and killing senescent cells, which are notorious for promoting aging, significant increases in lifespan and health are conceivable. 

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A New Form of Life is Discovered

An extremely short RNA is infectious and prevalent in the human microbiome.

While the last century might be called the DNA century, at least for molecular biology, the current century might be called that of RNA. A blizzard of new RNA types and potentials have been discovered in the normal eukaryotic milieu, including miRNA, eRNA, lincRNA. An RNA virus caused a pandemic, which was remedied by an RNA vaccine. Nobel prizes have been handed out in these fields, and we are also increasingly aware that RNA lies at the origin of life itself, as the first genetic and catalytic mechanism.

One of these Nobel prize winners recently undertook a hunt for small RNAs that might be lurking in the human microbiome- the soup of bacteria, fungi, and all the combined products that cover our surfaces, inside and out. What they found was astonishing- an RNA of merely 1164 nucleotides, which folds up into a rigid, linear rod, which they call "obelisks". This is not a product of the host genome, nor of any other known organism, but is rather some kind of extremely minimal pathogen that, like a transposon or self-splicing intron, is entirely nucleic-acid based. And the more they hunted, the more they found, ultimately finding thousands of obelisk-like entities hidden in the many databases of the world drawn from various environmental and microbiome samples. There is some precedent for this kind of structure, in the form of hepatitis D. This "viroid" of only 1682 nucleotides is a parasite of hepatitis B virus, depending on that virus for key replication functions. While normal viruses (like hepatitis B) encode many key functions of their own, like envelope proteins, genome packaging proteins, and replication enzymes, viroids tend to not encode anything, though hepatitis D does encode one antigenic protein, which exacerbates hepatitis B infections.

The obelisk RNA viroid-like species appear to encode one or two proteins, and possibly a ribozyme as well. The functions of all these are as yet unknown, but necessarily the RNAs rely entirely some host cell (currently unknown) functions to do their thing, such as the RNA polymerase to create copies of itself. Unknown also is whether they are dependent on other viruses, or only on cells for their propagation. Being just discovered, the researchers can do a great deal of bioinformatics, such as predicting the structure of the encoded protein, and the structure of the RNA genome. But key biology, like how they interact with host cells, what functions the host provides, and how they replicate, not to mention possible pathogenic consequences, remain unknown.

The highly self-complementary structure of one obelisk RNA sequence, leading to its identification and naming. In green is one reading frame, which codes for the main protein, of unknown function.

The curious thing about these new obelisk viroid-like RNAs is that, while common in human microbiomes, both oral and gut-derived, they are found only in 5-10% of them, not in all samples. This sort of suggests that they may account for some of the variability traceable to microbiomes, such as autoimmune issues, chronic ailments, nutritional variations, even effects on mood, etc.

Once a lot of databases were searched, obelisk RNAs turn up everywhere, even in some bacteria.

This work was done entirely in silico. Not a single wet-lab experiment was performed. It is a testament to the power of having alot of genomes at our disposal, and of modern computational firepower. This lab just had the idea that novel small viroid-like RNAs might exhibit certain types of (circular, self-complementary) structure, which led to this discovery of a novel form of "life". Are these RNAs alive? Certainly not. They are mere molecules and parasites that feed off, and transport themselves between, more fully functional cells. But they are part of the tapestry of life, which itself is wholly molecular, with many amazing emergent properties. Whether these obelisks turn out to have any medical or ecological significance, they are one more example of the lengths (and shorts) to which Darwinian selection has gone in the struggle for existence. 

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To Sleep- Perchance to Inactivate OX2R

The perils of developing sleeping, or anti-sleeping, drugs.

Sleep- the elixir of rest and repose. While we know of many good things that happen during sleep- the consolidation of memories, the cardiovascular rest, the hormonal and immune resetting, the slow waves and glymphatic cleansing of the brain- we don't know yet why it is absolutely essential, and lethal if repeatedly denied. Civilized life tends to damage our sleep habits, given artificial light and the endless distractions we have devised, leading to chronic sleeplessness and a spiral of narcotic drug consumption. Some conditions and mutations, like narcolepsy, have offered clues about how sleep is regulated, which has led to new treatments, though to be honest, good sleep hygiene is by far the best remedy.

Genetic narcolepsy was found to be due to mutations in the second receptor of the hormone orexin (OX2R), or also due to auto-immune conditions that kill off a specialized set of neurons in the hypothalamus- a basal part of the brain that sits just over the brain stem. This region normally has ~ 50,000 neurons that secrete orexin (which comes in two kinds as well, 1 and 2), and project to areas all over the brain, especially basal areas like the basal forebrain and amygdala, to regulate not just sleep but feeding, mood, reward, memory, and learning. Like any hormone receptor, the orexin receptors can be approached in two ways- by turning them on (agonist) or by turning them off (antagonist). Antagonist drugs were developed which turn off both orexin receptors, and thus promote sleep. The first was named suvorexant, using the "orex" and "ant" lexical elements to mark its functions, which is now standard for generic drug names. 

 This drug is moderately effective, and is a true sleep enhancer, promoting falling to sleep, restful sleep, and length of sleep, unlike some other sleep aids. Suvorexant antagonizes both receptors, but the researchers knew that only the deletion of OX2R, not OX1R, (in dogs, mice, and other animals), generates narcolepsy, so they developed a drug more specific to OX2R only. But the result was that it was less effective. It turned out that binding and turning off OX1R was helpful to sleep promotion, and there were no particularly bad side effects from binding both receptors, despite the wide ranging activities they appear to have. So while the trial of Merck's MK-1064 was successful, it was not better than their exising two-receptor drug, so its development was shelved. And we learned something intriguing about this system. While all animals have some kind of orexin, only mammals have the second orexin family member and receptor, suggesting that some interesting, but not complete, bifurcation happened in the functions of this system in evolution. 

What got me interested in this topic was a brief article from yet another drug company, Takeda, which was testing an agonist against the orexin receptors in an effort to treat narcolepsy. They created TAK-994, which binds to OX2R specifically, and showed a lot of promise in animal trials. It is a pill form, orally taken drug, in contrast to the existing treatment, danavorexton, which must be injected. In the human trial, it was remarkably effective, virtually eliminating cataleptic / narcoleptic episodes. But there was a problem- it caused enough liver toxicity that the trial was stopped and the drug shelved. Presumably, this company will try again, making variants of this compound that retain affinity and activity but not the toxicity. 

This brings up an underappreciated peril in drug design- where drugs end up. Drugs don't just go into our systems, hopefully slipping through the incredibly difficult gauntlet of our digestive system. But they all need to go somewhere after they have done their jobs, as well. Some drugs are hydrophilic enough, and generally inert enough, that they partition into the urine by dilution and don't have any further metabolic events. Most, however, are recognized by our internal detoxification systems as foreign, (that is, hydrophobic, but not recognizable as fats/lipids that are usual nutrients), and are derivatized by liver enzymes and sent out in the bile. 

Structure of TAK-994, which treats narcolepsy, but at the cost of liver dysfunction.

As you can see from the chemical structure above, TAK-994 is not a normal compound that might be encountered in the body, or as food. The amino sulfate is quite unusual, and the fluorines sprinkled about are totally unnatural. This would be a red flag substance, like the various PFAS materials we hear about in the news. The rings and fluorines create a relatively hydrophobic substance, which would need to be modified so that it can be routed out of the body. That is what a key enzyme of the liver, CYP3A4 does. It (and many family members that have arisen over evolutionary time) oxidizes all manner of foreign hydrophobic compounds, using a heme cofactor to handle the oxygen. It can add OH- groups (hydroxylation), break open double bonds (epoxidation), and break open phenol ring structures (aromatic oxidation). 

But then what? Evolution has met most of the toxic substances we meet with in nature with appropriate enzymes and routes out of the body. But these novel compounds we are making with modern chemistry are something else altogether. Some drugs are turned on by this process, waiting till they get to the liver to attain their active form. Others, apparently such as this one, are made into toxic compounds (as yet unknown) by this process, such that the liver is damaged. That is why animal studies and safety trials are so important. This drug binds to its target receptor, and does what it is supposed to do, but that isn't enough to be a good drug. 

 

• Crime in Connecticut.
• Some classic Bowie/Queen.

A Hard Road to a Cancer Drug

The long and winding story of the oncogene KRAS and its new drug, sotorasib.

After half a century of the "War on Cancer", new treatments are finally straggling into the clinic. It has been an extremely hard and frustrating road to study cancer, let alone treat it. We have learned amazing things, but mostly we have learned how convoluted a few billion years of evolution can make things. The regulatory landscape within our cells is undoubtedly the equal of any recalcitrant bureaucracy, full of redundant offices, multiple veto points, and stakeholders with obscure agendas. I recently watched a seminar in the field, which discussed one of the major genes mutated in cancer and what it has taken to develop a treatment against it. 

Cancer is caused by DNA mutations, and several different types need to occur in succession. There are driver mutations, which are the first step in the loss of normal cellular control. But additional mutations have to happen for such cells to progress through regulatory blocks, like escape from local environmental controls on cell type and cell division, past surveillance by the immune system, and past the reluctance of differentiated cells to migrate away from their resident organ. By the end, cancer cells typically have huge numbers of mutations, having incurred mutations in their DNA repair machinery in an adaptive effort to evade all these different controls.

While this means that many different targets exist that can treat some cancers, it also means that any single cancer requires a precisely tailored treatment, specific to its mutated genes. And that resistance is virtually inevitable given the highly mutable nature of these cells. 

One of the most common genes to be mutated to drive cancer (in roughly 20% of all cases) is KRAS, part of the RAS family of NRAS, KRAS, and HRAS. These were originally discovered through viruses that cause cancer in rats. These viruses (such as Kirsten rat sarcoma virus) had a copy of a rat gene in it, which it overpoduces and uses to overcome normal proliferation controls during infection. The viral gene was called an oncogene, and the original rat (or human) version was called a proto-oncogene, named KRAS. The RAS proteins occupy a central part of the signaling path that external events and stresses turn on to activate cell growth and proliferation, called the MAP kinase cascade. For instance, epidermal growth factor comes along in the blood, binds to a receptor on the outside of a cell, and turns on RAS, then MEK, MAPK, and finally transcription regulators that turn on genes in the nucleus, resulting in new proteins being expressed. "Turning on" means different things at each step in this cascade. The transcription regulators typically get phosphorylated by their upstream kinases like MAPK, which tag them for physical transport into the nucleus, where they can then activate genes. MAPK is turned on by being itself phosphorylated by MEK, and MEK is phosphorylated by RAF. RAF is turned on by binding to RAS, whose binding activity in turn is regulated by the state of a nucleotide (GTP) bound by RAS. When binding GTP, RAS is on, but if binding GDP, it is off.

A schematic of the RAS pathway, whereby extracellular growth signals are interpreted and amplified inside our cells, resulting in new gene expression as well as other more immediate effects. The cell surface receptor, activated by its ligand, activates associated SOS which activates RAS to the active (GTP) state. This leads to a kinase cascade through RAF, MEK, and MAPK and finally to gene regulators like MYC.

This whole system seems rather ornate, but it accomplishes one important thing, which is amplification. One turned-on RAF molecule or MEK molecule can turn on / phosphorylate many targets, so this cascade, though it appears linear in a diagram, is acutally a chain reaction of sorts, amplifying as it goes along. And what governs the state of RAS and its bound GTP? The state of the EGFR receptor, of course. When KRAS is activated, the resident GDP leaves, and GTP comes to take its place. RAS is a weak GTPase enzyme itself, slowly converting itself from the active back to the inactive state with GDP. 

Given all this, one would think that RAS, and KRAS in particular, might be "druggable", by sticking some well-designed molecule into the GTP/GDP binding pocket and freezing it in an inactive state. But the sad fact of the matter is that the affinity KRAS has to GTP is incredibly high- so high it is hard to measure, with a binding constant of about 20 pM. That is, half the KRAS-bound GTP comes off when the ambient concentration of GTP is infinitesimal, 0.02 nano molar. This means that nothing else is likely to be designed that can displace GTP or GDP from the KRAS protein, which means that in traditional terms, it is "undruggable". What is the biological logic of this? Well, it turns out that the RAS enzymes are managed by yet other proteins, which have the specific roles of prying GDP off (GTP exchange factor, or GEF) and of activating the GTP-ase activity of RAS to convert GTP to GDP (GTPase activating protein, or GAP). It is the GEF protein that is stimulated by the receptors like EGFR that induce RAS activity. 

So we have to be cleverer in finding ways to attack this protein. Incidentally, most of the oncogenic mutations of KRAS are at the twelfth residue, glycine, which occupies a key part of the GAP binding site. As glycine is the smallest amino acid, any other amino acid here is bulkier, and blocks GAP binding, which means that KRAS with any of these mutations can not be turned off. It just keeps on signaling and signaling, driving the cell to think it needs to grow all the time. This property of gain of function and the ability of any mutation to fit the bill is why this particular defect in KRAS is such a common cancer-driving mutation. It accounts for ~90% of pancreatic cancers, for instance. 

The seminar went on a long tangent, which occupied the field (of those looking for ways to inhibit KRAS with drugs) for roughly a decade. RAS proteins are not intrinsically membrane proteins, but they are covalently modified with a farnesyl fatty tail, which keeps them stuck in the cell's plasma membrane. Indeed, if this modification is prevented, RAS proteins don't work. So great- how to prevent that? Several groups developed inhibitors of the farnesyl transferase enzyme that carries out this modification. The inhibitors worked great, since the farnesyl transferase has a nice big pocket for its large substrate to bind, and doesn't bind it too tightly. But they didn't inhibit the RAS proteins, because there was a backup system- geranygeranyl transferase that steps into the breach as a backup, which can attach an even bigger fatty tail to RAS proteins. Arghhh!

While some are working on inhibiting both enzymes, the presenter, Kevan Shokat of UCSF, went in another direction. As a chemist, he figured that for the fraction of the KRAS mutants at position 12 that transform from glycine to cysteine, some very specific chemistry (that is, easy methods of cross-linking), can be brought to bear. Given the nature of the genetic code, the fraction of mutations that go from glycine to cysteine are small, there being eight amino acids that are within a one-base change of glycine, coded by GGT. So at best, this approach is going to have a modest impact. Nevertheless, there was little choice, so they forged ahead with a complicated chemical scheme to make a small molecule that could chemically crosslink to that cysteine, with selectivity determined by a modest shape fit to the surface of the KRAS protein near this GEF binding site. 

A structural model of KRAS, with its extremely tightly-bound substrate GDP in orange. The drug sotorasib is below in teal, bound in another pocket, with a tail extending upwards to the (mutant) cysteine 12, which is not differentiated by color, but sits over a magnesium ion (green) being coordinated by GDP. The main job of sotorasib is to interfere with the binding of the guanine exchange factor (GEF) which happens on the surface to its left, and would reset KRAS to an active state.

This approach worked surprisingly well, as the KRAS protein obligingly offfered a cryptic nook that the chemists took advantage of to make this hybrid compound, now called the drug sotorasib. This is an FDA-approved treatment for cancers which are specifically driven by this particular KRAS mutation of position 12 from glycine to cysteine. That research group is currently trying to extend their method to other mutant forms, with modest success. 

So let's take a step back. This new treatment requires, obviously, the patient's tumor to be sequenced to figure out its molecular nature. That is pretty standard these days. And then, only a small fraction of patients will get the good news that this drug may help them. Lung cancers are the principal candidates currently, (of which about 15% have this mutation), while only about 1-2% of other cancers have this mutation. This drug has some toxicity- while it is a magic bullet, its magic is far from perfect, (which is odd given the exquisite selectivity it has for the mutated form of KRAS, which should only exist in cancer tissues). And lastly, it gives, on average, under six months of reprieve from cancer progression, compared to four and a half months with a more generic drug. As mentioned above, tumors at this stage are riven with other mutations and evolve resistence to this treatment with appalling relentlessness.

While it is great to have developed a new class of drugs like this one against a very recalcitrant target, and done so on a highly rational basis driven by our growing molecular knowlege of cancer biology, this result seems like a bit of a let-down. And note also that this achievement required decades of publicly funded research, and doubtless a billion dollars or more of corporate investment to get to this point. Costs are about twenty five thousand dollars per patient, and overall sales are maybe two hundred million dollars per year, expected to increase steadily.

Does this all make sense? I am not sure, but perhaps the important part is that things can not get worse. The patent on this drug will eventually expire and its costs will come down. And the research community will keep looking for other, better ways to attack hard targets like KRAS, and will someday succeed.

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How Recessive is a Recessive Mutation?

Many relationships exist between mutation, copy number, and phenotype.

The traditional setup of Mendelian genetics is that an allele of a gene is either recessive or dominant. Blue eyes are recessive to brown eyes, for the simple reason that blue arises from the absence of an enzyme, due to a loss of function mutation. So having some of that enzyme, from even one "brown" copy of that gene, is dominant over the defective "blue" copy. You need two "blue" alleles to have blue eyes. This could be generalized to most genes, especially essential genes, where lacking both copies is lethal, while having one working copy will get you through, and cover for a defective copy. Most gene mutations are, by this model, recessive. 

But most loci and mutations implicated in disease don't really work like that. Some recent papers delved into the genetics of such mutations, and observed that their recessiveness was all over the map, a spectrum, really, of effects from fully recessive to dominant, with most in the middle ground. This is informative for clinical genetics, but also for evolutionary studies, suggesting that evolution is not, after all, blind to the majority of mutations, which are mostly deleterious, exist most of the time in the haploid (one-copy) state, and would be wholly recessive by the usual assumption.

The first paper describes a large study over the Finnish population, which benefited from several advantages. Finns have a good health system with thorough records which are housed in a national biobank. The study used 177,000 health records and 83,000 variants in coding regions of genes collected from sequencing studies. Second, the Finnish population is relatively small and has experienced bottlenecks from smaller founding populations, which amplifies the prevalence of variants that those founders had. That allows those variants to rise to higher rates of appearance, especially in the homozygous state, which generally causes more noticeable disease phenotypes. Both the detectability and the statistics were powered by this higher incidence of some deleterious mutations (while others, naturally, would have been more rare than the world-wide average, or absent altogether).

Thirdly, the authors emphasize that they searched for various levels of recessive effect, which is contrary to the usual practice of just assuming a linear effect. A linear model says that one copy of a mutation has half the effect of two copies- which is true sometimes, but not most of the time, especially in more typical cases of recessive effect where one copy has a good deal less effect, if not zero. Returning to eye color, if one looks in detail, there are many shades of eyes, even of blue eyes, so it is evident that the alleles that affect eye color are various, and express to different degrees (have various penetrance, in the parlance). While complete recessiveness happens frequently, it is not the most common case, since we generally do not routinely express excess amounts of proteins from our genes, making loss of one copy noticeable most of the time, to some degree. This is why the lack of a whole chromosome, or an excess of a whole chromosome, has generally devastating consequences. Trisomies in only three chromosomes are viable (that is, not lethal), and confer various severe syndromes.

A population proportion plot vs age of disease diagnosis for three different diseases and an associated genetic variant. In blue is the normal ("wild-type") case, in yellow is the heterozygote, and in red the homozygote with two variant alleles. For "b", the total lack of XPA causes skin cancer with juvenile onset, and the homozygotic case is not shown. The Finnish data allowed detection of rather small recessive effects from variations that are common in that population. For instanace, "a" shows the barely discernable advancement of age of diagnosis for a disease (hearing loss) that in the homozygotic state is universal by age 10, caused by mutations in GJB2.

The second paper looked more directly at the fitness cost of variations over large populations, in the heterozygous state. They looked at loss-of-function (LOF) mutations of over 17,000 genes, studying their rate of appearance and loss from human populations, as well as in pedigrees. These rates were turned, by a modeling system, into fitness costs, which are stated in percentage terms, vs wild type. A fitness cost of 1% is pretty mild, (though highly significant over longer evolutionary time), while a fitness cost of 10% is quite severe, and one of 100% is immediately lethal and would never be observed in the population. For example, a mutation that is seen rarely, and in pedigrees only persists for a couple of generations, implies a fitness cost of over 10%.

They come up with a parameter "hs", which is the fitness cost "s" of losing both copies of a gene, multiplied by "h", a measure of the dominance of the mutation in a single copy.

In these graphs, human genes are stacked up in the Y axis sorted by their computed "hs" fitness cost in the heterozygous state. Error bars are in blue, showing that this is naturally a rather error-prone exercise of estimation. But what is significant is that most genes are somewhere on the spectrum, with very few having negligible effects, (bottom), and many having highly significant effects (top). Genes on the X chromosome are naturally skewed to much higher significance when mutated, since in males there is no other copy, and even in females, one X chromosome is (randomly) inactivated to provide dosage compensation- that is, to match the male dosage of production of X genes- which results in much higher penetrance for females as well.

So the bottom line is that while diploidy helps to hide alot of variation in sexual organisms, and in humans in particular, it does not hide it completely. We are each estimated to receive, at birth, about 70 new mutations, of which 1/1000 are the kind of total loss of gene function studied here. This work then estimates that 20% of those mutations have a severe fitness effect of >10%, meaning that about one in seventy zygotes carry such a new mutation, not counting what it has inherited from its parents, and will suffer ill effects immediately, even though it has a wild-type copy of that gene as well.

Humans, as other organisms, have a large mutational load that is constantly under surveillance by natural selection. The fact that severe mutations routinely still have significant effects in the heterozygous state is both good and bad news. Good in the sense that natural selection has more to work with and can gradually whittle down on their frequency without necessarily waiting for the chance of two meeting in an unfortunate homozygous state. But bad in the sense that it adds to our overall phenotypic variation and health difficulties a whole new set of deficiencies that, while individually and typically minor, are also legion.

• Psychological projection, to the max.
• And another murder.
• Next up, book burnings.
• Medicare advantage is a corrupt disaster.
• Corporate health insurance says no.

Senescent, Cancerous Cannibals

Tumor cells not only escape normal cell proliferation controls, but some of them eat nearby cells.

Our cells live in an uneasy truce. Cooperation is prized and specialization into different cell types, tissues, and organs is pervasive. But deep down, each cell wants to grow and survive, prompting many mechanisms of control, such as cell suicide (apoptosis) and immunological surveillance (macrophages, killer T-cells). Cancer is the ultimate betrayal, not only showing disregard for the ruling order, but in its worst forms killing the whole organism in a pointless drive for growth.

A fascinating control mechanism that has come to prominence recently is cellular senescence. In petri dishes, cells can only be goosed along for a few dozen cycles of division until they give out, and become senescent. Which is to say, they cease replicating but remain alive. It was first thought that this was another mechanism to keep cancer under control, restricting replication to "stem" cells and their recent progeny. But a lot of confusing and interesting observations indicate that the deeper meaning of senescence lies in development, where it appears to function as an alternate form of cell suicide, delayed so that tissues are less disrupted. 

Apoptosis is used very widely during development to reshape tissues, and senescence is used extensively as well in these programs. Senescent cells are far from quiescent, however. They have high metabolic activity and are particularly notorious for secreting a witches' brew of inflammatory cytokines and other proteins- the senescence-associated secretory phenotype, or SASP. in the normal course of events, this attracts immune system cells which initiate repair and clearance operations that remove the senescent cells and make sure the tissue remains on track to fulfill its program. These SASP products can turn nearby cells to senescence as well, and form an inflammatory micro-environment that, if resolved rapidly, is harmless, but if persistent, can lead to bad, even cancerous local outcomes. 

The significance of senescent cells has been highlighted in aging, where they are found to be immensely influential. To quote the wiki site:

"Transplantation of only a few (1 per 10,000) senescent cells into lean middle-aged mice was shown to be sufficient to induce frailty, early onset of aging-associated diseases, and premature death."

The logic behind all this seems to be another curse of aging, which is that while we are young, senescent cells are cleared with very high efficiency. But as the immune system ages, a very small proportion of senescent cells are missed, which are, evolutionarily speaking, an afterthought, but gradually accumulate with age, and powerfully push the aging process along. We are, after all, anxious to reduce chronic inflammation, for example. A quest for "senolytic" therapies to clear senescent cells is becoming a big theme in academia and the drug industry and may eventually have very significant benefits. 

Another odd property of senescent cells is that their program, and the effects they have on nearby cells, resemble to some partial degree those of stem cells. That is, the prevention of cell death is a common property, as is the prevention of certain controls preventing differentiation. This brings us to tumor cells, which frequently enter senescence under stress, like that of chemotherapy. This fate is highly ambivalent. It would have been better for such cells to die outright, of course. Most senescent tumor cells stay in senescence, which is bad enough for their SASP effects in the local environment. But a few tumor cells emerge from senescence, (whether due to further mutations or other sporadic properties is as yet unknown), and they do so with more stem-like character that makes them more proliferative and malignant.

A recent paper offered a new wrinkle on this situation, finding that senescent tumor cells have a novel property- that of eating neighboring cells. As mentioned above, senescent cells have high metabolic demands, as do tumor cells, so finding enough food is always an issue. But in the normal body, only very few cells are empowered to eat other cells- i.e. those of the immune system. To find other cells doing this is highly unusual, interesting, and disturbing. It is one more item in the list of bad things that happen when senescence and cancer combine forces.

A senescent tumor cell (green) phagocytoses and digests a normal cell (red).

• Shockingly, some people are decent.
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• Keep cats indoors.

Death at the Starting Line- Aneuploidy and Selfish Centromeres

Mammalian reproduction is unusually wasteful, due to some interesting processes and tradeoffs.

Now that we have settled the facts that life begins at conception and abortion is murder, a minor question arises. There is a lot of murder going on in early embryogenesis, and who is responsible? Probably god. Roughly two-thirds of embryos that form are aneuploid (have an extra chromosome or lack a chromosome) and die, usually very soon. Those that continue to later stages of pregnancy cause a high rate of miscarriages-about 15% of pregnancies. A recent paper points out that these rates are unusual compared with most eukaryotes. Mammals are virtually alone in exhibiting such high wastefulness, and the author proposes an interesting explanation for it.

First, some perspective on aneupoidy. Germ cells go through a two-stage process of meiosis where their DNA is divided two ways, first by homolog pairs, (that is, the sets inherited from each parent, with some amount of crossing-over that provides random recombination), and second by individual chromosomes. In more primitive organisms (like yeast) this is an efficient, symmetrical, and not-at-all wasteful process. Any loss of genetic material would be abhorrent, as the cells are putting every molecule of their being into the four resulting spores, each of which are viable.

A standard diagram of meiosis. Note that the microtubules (yellow) engage in a gradual and competitive process of capturing centromeres of each chromosome to arrive at the final state of regular alignment, which can then be followed by even division of the genetic material and the cell.

In animals, on the other hand, meiosis of egg cells is asymmetric, yielding one ovum / egg and three polar bodies, which  have various roles in some species to assist development, but are ultimately discarded. This asymmetric division sets up a competition between chromosomes to get into the egg, rather than into a polar body. One would think that chromosomes don't have much say in the matter, but actually, cell division is a very delicate process that can be gamed by "strong" centromeres.

Centromeres are the central structures on chromosomes that form attachments to the microtubules forming the mitotic spindle. This attachment process is highly dynamic and even competitive, with microtubules testing out centromere attachment sites, and using tension ultimately as the mark of having a properly oriented chromosome with microtubules from each side of the dividing cell (i.e. each microtubule organizing center) attached to each of the centromeres, holding them steady and in tension at the midline of the cell. Well, in oocytes, this does not happen at the midline, but lopsidedly towards one pole, given that one of the product cells is going to be much larger than the others. 

In oocytes, cell division is highly asymmetric with a winner-take-all result. This opens the door to a mortal competition among chromosomes to detect which side is which and to get on the winning side. 

One of the mysteries of biology is why the centromere is a highly degenerate, and also a speedily evolving, structure. They are made up of huge regions of monotonously repeated DNA, which have been especially difficult to sequence accurately. Well, this competition to get into the next generation can go some way to explain this structure, and also why it changes rapidly, (on evolutionary time scales), as centromeric repeats expand to capture more microtubules and get into the egg, and other portions of the machinery evolve to dampen this unsociable behavior and keep everyone in line. It is a veritable arms race. 

But the funny thing is that it is only mammals that show a particularly wasteful form of this behavior, in the form of frequent aneuploidy. The competition is so brazen that some centromeres force their way into the egg when there is already another copy there, generating at best a syndrome like Down, but for all other chromosomes than #21, certain death. This seems rather self-defeating. Or does it?

The latest paper observes that mammals devote a great deal of care to their offspring, making them different from fish, amphibians, and even birds, which put most of their effort into producing the very large egg, and relatively less (though still significant amounts) into care of infants. This huge investment of resources means that causing a miscarriage or earlier termination is not a total loss at all, for the rudely trisomic extra chromosome. No, it allows resource recovery in the form of another attempt at pregnancy, typically quite soon thereafter, at which point the pushy chromosome gets another chance to form a proper egg. It is a classic case of extortion at the molecular scale. 

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