Asparaginase therapy for ALL, biochemistry of asparaginase and asparagine synthetase

Asparagine biochemistry, asparagine synthetase, and asparaginase treatment for ALL.You never know where a scientific exploration will take you - I learned so much about so many different things just by researching single amino acids, and Asparagine (Asn, N) turns out to be one of those story-starters. Yes, the name does come from asparagus, but that’s not the big story I want to tell you about. Today is ALL about Asn & how childhood Acute Lymphoblastic Leukemia (ALL) has an “Achille’s heel” for it that is targeted in mainstay treatment.

It’s Day 17 of #20DaysOfAminoAcids - the bumbling biochemist’s version of an advent calendar. Amino acids are the building blocks of proteins. There are 20 (common) genetically-specified ones, each with a generic backbone with to allow for linking up through peptide bonds to form chains (polypeptides) that fold up into functional proteins, as well as unique side chains (aka “R groups” that stick off like charms from a charm bracelet). Each day I’m going to bring you the story of one of these “charms” - what we know about it and how we know about it, where it comes from, where it goes, and outstanding questions nobody knows.

Those generic parts are attached to a central “alpha carbon” (Ca), which is also attached to one of 20 unique side chains (“R groups”) which have different properties (big, small, hydrophilic (water-loving), hydrophobic (water-avoided), etc.) & proteins have different combos of them, so the proteins have different properties. And we can get a better appreciation and understanding of proteins if we look at those letters. So, today let’s look at Asparagine (Asn, N).

Unlike Asp, which is negatively-charged under usual bodily (physiological) conditions, asparagine is neutral. But they look a lot alike - and as we’ll see, our bodies can convert between the two of them. Both have a methylene (-CH₂) followed by a carbonyl (C=O) in their side chain, but they differ in what’s attached to that carbonyl C on the other side. Asp has another O, giving it a “carboxyl group” but Asn has an amino group (-NH₂) instead. This makes Asn the AMIDE of Asp. AM I DEscribing something without defining the jargon? I’ll fix that - and tell you more about Asn biochemistry - after I tell you a story about it (don’t worry, you don’t need to know the nomenclature stuff for it)).

Asparagine is considered nonessential in the dietary sense of the word - our bodies can make it. But they can also break it, and they can use it to build other molecules (this can be joining up with other peptides to form big ole proteins, or getting modified to form other small molecules). So the levels of free Asn in your body at any time depend on how much of it you eat, how much you make, how much you break, & how much you “siphon away.” Your body keeps a nice balance by regulating the making, breaking, and other-purpose-using.

In a type of blood cancer called childhood Acute Lymphoblastic Leukemia (ALL), the balance gets thrown off because the cells lose their ability to make it as much, but they still need it, so they rely on a steady source of “pre-made” Asn from the bloodstream. This has led to a therapeutic strategy of depriving these cells of what little Asn they do have by adding extra “breakers” into the bloodstream, depleting the Asn supply - this strategy selectively affects the cancer cells because the other cells have enough “makers” to counterbalance these “breakers”

So let’s meet these molecular workers, which have the general job description of “enzymes.” An enzyme is a reaction-speed-upper - it’s usually a protein, sometimes a protein/RNA combo, sometimes RNA alone, and it helps mediate reactions by doing things like bringing reactants together and holding them in the right orientation with the right ambience, etc. You know you’re probably dealing with an enzyme if you see the ending “-ase”

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[thebumblingbiochemist]

The “maker” is an enzyme called Asparagine Synthetase (ASNS) and it makes Asn through a “transamination” reaction which transfers an amino group from the amino acid glutamine to aspartate, turning aspartate into asparagine and glutamine into glutamate. It has a pretty cool reaction - I won’t bore you too much, but it has a mechanism involving 2 “active sites” (places where reactions occur). It requires spending a molecule of ATP to “activate” the carboxylate group in Asp’s side chain that you want to turn into an amide. First the carboxylate attacks ATP’s innermost phosphate group, kicking off 2 of the phosphates as inorganic (non-carbon-attached) pyrophosphate (PPi), and leaving you with a β-aspartyl-AMP intermediate where Asp has AMP stuck to it as a big ole, energetically unstable, “attack here” sign. Meanwhile, in the enzyme’s second active site, the amine group is cut off from glutamine as ammonia (NH₃), which travels through a secret tunnel between the active sites to attack, releasing AMP & swapping it out for an amino group.

The “breaker” is an enzyme called Asparaginase (ASNase). It catalyzes the splitting up of Asn into aspartate and ammonia. When Asn is broken down for energy, it follows the usual “remove amino parts & process them and the carbon skeletons separately” routine we’ve seen. But, unlike most of the other amino acids, we have 2 amino groups to remove. ASNase does the first removal - that of the “extra” amino group - the side chain one, to give you aspartate. And then that aspartate gets broken down the same way aspartate does - because it *is* aspartate now! The backbone amino group is removed by aspartate aminotransferase to give you OXALOACETATE, which is nitrogen-free and can get processed in the citric acid cycle (aka TCA, aka Krebs cycle) to get energy. Alternatively, the Asp can be used as Asp.

It’s the ASNase that’s used as an anti-cancer treatment - and the version that’s used as a drug comes from bacteria - as a biochemist, I’m really used to the concept of using bacterial proteins to do things for us - like using bacterial “restriction enzymes” (site-specific endonucleases) to recognize and cut specific DNA sequences. When I’m using bacterial proteins, it’s in a test tube, (in vitro) but turns out bacterial enzymes can be used in people too (in vivo). Including to treat childhood Acute Lymphoblastic Leukemia.

“Leukemia” is an umbrella term for cancers that affect blood & bone marrow (where blood cells are made). There are different types of leukemia that affect different types of blood cells - ALL affects lymphoid progenitor cells (a type of white blood cell important for the immune system) and can cause problems including anemia, fatigue, and immune system dysfunction. First tried out in the 1960s (more on the history below), E. coli ASNase has become a mainstay of treatment for childhood ALL - in addition to traditional chemotherapy drugs like vincristine & prednisone, patients are given this enzyme, and here’s why it *hopefully* works.

Both normal and cancer lymphocytes need Asn - and they get their supply by taking it in from circulating blood plasma (plasma is just the non-cell part of the blood). When you introduce ASNase into the blood, it goes to work breaking the Asn down, depleting this supply. This causes leukemic blasts to actually start shipping their cytoplasmic stock out (which gets broken down), so they get depleted intracellularly. And, unlike normal cells, these cancer cells aren’t able to make much if any of their own because, for reasons still not fully understood, they don’t make much of the “maker, ” ASNS. But they need Asn to build proteins and survive - and now they can’t get it from the blood & they can’t make it, so they die off. But the normal cells are fine because they *do* express ASNS & therefore they can make enough to compensate for what they can’t get “pre-made” from the bloodstream.

ASNase also depletes the glutamine pool because, even though ASNase prefers breaking down Asn, it can also deaminate glutamine (into glutamate & ammonia). So now you’re messing with levels of multiple amino acids. One of the coolest things about metabolism is that it’s like the “7 degrees to Kevin Bacon” thing - all molecules seem to be somehow connected. But the non-cool thing about this is that messing up one or a couple of the players (especially central hub ones) can send the whole metabolism out of whack. So, among other things, ASNase treatment can lead to global metabolic changes with less glycolysis (sugar breakdown) and increased use of fats for energy. Sensing amino acid deprivation, cells shut down protein-making and even activate autophagy (selective breakdown of intracellular parts) & apoptosis (programmed cell death).

But cancer cells are tricky. Because they bypass traditional cellular “safety checks, ” they can grow and divide quickly and without adequate proofreading of the DNA they copy. This allows for evolution on the really small scale - cells that randomly happen to acquire mutations that help give them a growth and/or survival advantage will out-compete other cells, so if a tumor cell happens to get a random mutation that decreases its reliance on Asn, it’ll thrive and multiply, leading to drug-resistant cancer.


The history of the ASNase/leukemia link goes back to the 1950s - In 1953, Kidd found that guinea pig serum caused regression of implanted lymphoma cells in mice. And then in follow-up studies, Broome found that the anti-lymphoma effect was coming from asparaginase. Dolowy et al. & Hill et al. tried ASNase treatment in human patients in the mid-1960s, but it didn’t really take off as treatment until the 1970s, when they were able to produce ASNase at larger scales so they had enough for large-scale trials. Those larger trials found that ASNase helped, so it has become a standard part of childhood ALL treatment, approved for medical use in the US in 1978, and on the WHO’s list of essential medicines. ASNase is also used for treating some forms of acute myeloblastic leukemia (AML).

So worldwide, you need a lot of it. Even those early trials were held up by supply - so scientists started looking for sources of lots of asparaginase to test. And bacteria are great for making lots of protein on the cheap (one of the reasons we often use it to recombinantly express proteins (stick in genes for proteins we want made and let them make it for us). But in this case, it’s the bacteria’s own “native” proteins that are isolated and used. E. coli is the main source of therapeutic ASNase and it can be injected into the muscles, under the skin, or through IV. It’s often given in a “PEGylated” form - it has polyethylene glycol (PEG) (carbon-carbon-oxygen chains) attached to it that help with stability & help hide it from the immune system.