Discovering New ALS Therapeutic Avenues with Evangelos Kiskinis, PhD

Episode Transcript
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Erin Spain, MS (00:10):
This is Breakthroughs, a podcast from Northwestern University
Feinberg School of Medicine.
I'm Erin Spain, host of the show.
New Northwestern Medicine studies aredeepening our understanding of ALS.
Or amyotrophic lateral sclerosis,a progressive neurodegenerative

(00:30):
disease that attacks motor neuronsand the brain and spinal cord.
This research from the lab of EvangelosKiskinis could lead to new avenues for
the development of targeted therapies.
Dr.
Kiskinis joins me todayto discuss his work.
He is an associate professor of neurologyin the division of neuromuscular
disease and of neuroscience at Feinberg.

(00:51):
Welcome to the show.

Evangelos Kiskinis, PhD: Very happy to be here. (00:53):
undefined
Thank you for having me.

Well, let's talk about ALS more than 32,000 Americans
are currently living with this deadlycondition, which is very difficult
to diagnose, manage, and treat.
Describe this condition to me andhow you're studying it in your lab.

Evangelos Kiskinis, PhD (01:11):
So ALS is really devastating neurodegenerative condition.
The patients unfortunately exhibita progressive inability to control
their muscles that leads to eventualcomplete paralysis, which becomes fatal.
Now, what's characteristic aboutthis disease is, while all of this
is happening in the majority ofpatients, your consciousness, your

(01:32):
brain is completely unaffected.
So it's also been describedas locked in syndrome.
Unfortunately, the current expectationof survival from diagnosis to eventual
death is between two to five years.
Not much time, so it's veryaggressive, it's vastly progressing.
The clinical presentation of ALS iscaused by the underlying dysfunction

(01:55):
and eventual degeneration of a veryparticular type of nerve cell in the
human body that's called the motor neuron.
And these motor neurons are the nervecells that essentially connect our brains
to our muscles and thus allow us to move,to walk, to speak, to swallow, to breathe,
and execute all autonomic functions.
For reasons that we do not quiteunderstand, people that get diagnosed

(02:17):
with this disease, these particularnerve cells progressively degenerate.

And tell me about the work in your lab to study this disease.
We should mention most people havea sporadic form of this disease,
but there is also a genetic form.
Tell me about that andspecifically how you're approaching
this condition in your lab.

The overwhelming majority of ALS patients are characterized
as suffering from sporadic disease.
All that that means is thatthere's no family history.
That represents about 90 percent of cases.
About 10 percent of cases run in families.
There's multiple family members inevery generation that get this disease.

(02:57):
What we know is that the familialcases are caused by very strong
highly penetrant genetic causes.
So these are mutations that arefound in particular genes that
basically will guarantee thatsomebody will get that disease.
That's what penetrance means.
And sporadic disease, the short answeris we don't know exactly what causes it,

(03:18):
but what we think is that it is likely acombination of genetic predisposition--
so people have maybe many mutations indifferent genes that interact with each
other-- as well as environmental factors.
We don't really understand whatdrives disease in sporadic cases.

(03:38):
Now I will say that one of thereasons we do not know a lot about
ALS is because historically it's beena very difficult disease to study.
The nerve cells, the motor nerves that areaffected in this disease and are driving
it, are within the central nervous system.
That means in our brains and our spinalcords, so they're really hard to access.

(03:59):
And the second issue is that,as we've said, the sporadic
disease is really hard to model.
Traditionally neuroscientistshave relied on animal models.
But unfortunately, because insporadic disease, it's probably
a combination of genes or genesinteracting with environmental
factors, that's been hard to model.
It's not amenable totraditional approaches.

(04:19):
So, what my lab does, what, what we'veproposed to do a few years ago at
this point was to use an alternativeapproach, which is based on induced
pluripotent stem cell technologies (iPSC).
Induced pluripotent stem cells areembryonic stem cells that we can
create from any given individual.
This is a fantastic achievement that wonthe Nobel Prize a few years ago by a very

(04:43):
famous scientist called Shina Yamanaka,who figured out that you could take any
sort of somatic cell from an individual,a somatic cell could be a skin cell, a
blood cell, and using a combination ofmolecular factors, you can turn those
somatic cells into cells that behavejust like embryonic stem cells . Really
fascinating, incredible discovery.

(05:05):
And embryonic stem cells have two uniquecharacteristics: A, they can generate
any cell type in the human body becauseat some point every one of us was just a
bunch of those human embryonic stem cells,and eventually they differentiate and then
they turn to different organs and tissues.
And B, under the right conditions,they can divide indefinite.

(05:27):
And that means that once wehave them, we have them forever.
We can make loads and loads of them,and we can coax them into making
distinct cell types that representthe human body, like nerve cells, like
the nerve cells that degenerate inALS patients, the motor neurons, by
applying to them a combination of eithersmall molecules or molecular factors.

(05:49):
This is really powerful because itallows us to make the motor neurons
from a patient that has ALS, and wecan study their motor neurons in the
lab while this patient is still around.

So you've been using this technology to model ALS for about 15
years, but since coming to Northwestern,about eight years ago, you've really been
laser focused on two overall objectives.
Tell me about that.

Ultimately, we're trying to understand whether
the molecular causes that drive thedysfunction of these nerve cells are
the same or different between thesedistinct genetic subtypes of ALS.
That's the primary goal.
And the secondary goal is, once wedefine the molecular causes in these
rare genetic subtypes of ALS, we cannow make iPSCs or induced pluripotent

(06:38):
stem cells from also spradic cases.
And then we can ask whether themechanisms that drive the degeneration
in these rare genetic subtypes arealso relevant in sporadic disease.
And as we discussed earlier, thisis the first time that we can
really model and go after sporadicdisease using these technologies.

And you've made some discoveries recently.
You've actually publishedthree papers since August 2023.
And I want to talk about some of thosepapers and what you've uncovered.
So let's start with the firstin Science Advances that was
published in August 2023.
Tell me about your discovery there.

That first paper that we published late in the summer
describes our efforts to understandthe causes of neuronal dysfunction and
eventual degeneration in one of thenewest discovered genetic causes of ALS.
So this is one of the newer genesthat was discovered a few years ago.

(07:34):
And actually, what's fascinatingabout this gene, the gene
is called NEK1, N-E-K-1.
The genetic discovery was largelyfunded by money that was raised
during the Ice Bucket Challenge.
The Ice Bucket Challenge is this amazingcultural phenomenon that started by
these two kids that said, well, wegot to raise awareness around ALS.

(07:55):
in the early, I guess, times ofsocial media, they had this brilliant
idea of getting a bucket of ice coldwater and throwing it on somebody.
The idea behind this is let's makean effort to sort of freeze ALS.
Let's like figure out a wayto make meaningful therapeutic
discoveries for these disease.
So people would donate, peoplewould challenge each other and it

(08:18):
became a social media phenomenon.
It raised a phenomenal amount offunds worldwide that were used
to support research around ALS.
One of the major genetic discoveriesthat came out of that effort is the
discovery of this gene called NEK1as a gene that can cause a disease.
Now what's fascinating about thisgene is that it is responsible

(08:41):
potentially for as much as 3 percentof all ALS, which I know sounds like
a small proportion, but when it comesto ALS, this is probably the third
biggest genetic cause of the disease.
So we got excited about that discovery,and to begin to address this question of
how does this, how do mutations of thisgene cause ALS, we made patient specific

(09:01):
induced pluripotent stem cells frompeople that had mutations in this gene.
We made nerve cells and we asked, usinga combination of approaches, why do
these nerve cells become dysfunctional?
And what we essentially uncoveredwas that NEK1 is a protein
that's known as a kinase.
And what kinases do is they phosphorylateother target proteins to modulate the

(09:25):
function of these target proteins.
So what phosphorylation meansis a modification, is a chemical
modification on a protein cellsutilize to change or modulate the
function of that particular protein.
So we discovered that thisNEK1 targets a number of other

(09:46):
proteins in human nerve cells.
And most of these proteins, to oursurprise, were involved in two fundamental
cellular pathways in nerve cells.
One of those pathways is knownas nucleocytoplasmic transport.
And all that that means is it's thepathway that regulates how proteins
and RNAs go in and out of the nucleus.

(10:08):
That's a very well safeguardedprocess, and there's particular
proteins that transport thingsin and out of the nucleus.
What we discovered was that NEK1phosphorylates or can phosphorylate
a number of these proteins.
And this was an exciting discoverybecause that particular pathway nuclear
import had recently been highlightedas a pathway that becomes dysfunctional

(10:32):
in other genetic subtypes of ALS.
So this provides an example of howtwo distinct genetic subtypes of ALS
meet or converge at targeting thesame molecular pathway in a nerve cell.
And that's the pathway of nuclearimport . Now, what we think is
happening is that nuclear importbecomes a little bit less efficient.

(10:55):
So fewer things that need to go intothe nucleus eventually make it there.
And as a result of that, thecells become dysfunctional.
So that was one of thepathways that we uncovered.
And the other one, which is just asexciting, is we found the NEK1 targets
for phosphorylation proteins known asmicrotubules, or I should say tubulins,
which assemble into microtubules.

(11:15):
And what microtubules are, they'rebasically the structural, the
building blocks of the cytoskeleton.
So the structure of most celltypes, including the nerve cell.
And what we uncovered is that the lackof effective NEK1 activity leads to
disruption of this microtubule network,which again is very exciting because

(11:36):
disruptions of the microtubule networkhave been previously highlighted as
another pathway that becomes dysfunctionalin ALS, including in sporadic disease.

And in this study, you were able to introduce anti-cancer drugs.
Tell me about that and howthat fits into the discovery.

Anti cancer drugs, this is a particular class of
anti cancer drugs that target thisparticular pathway, the microtubule
pathway, because if cancer cells,which divide all the time, cannot
properly control the microtubules,they stop dividing, they degenerate.
So it turns out that in ALSneurons, this pathway is

(12:16):
disrupted but in the opposite way.
So it turns out that in ALS,nerve cells from ALS patients,
the microtubules are less stable.
And we thought, well, if we applythese anti cancer drugs that
stabilize microtubules, that couldbe a good thing in a nerve cell which
doesn't divide, doesn't replicate,so it doesn't need to disassemble

(12:37):
and reassemble its microtubules.
And indeed, when we applied these drugs,we're able to stabilize microtubules and
then patient nerve cells did a lot better.
Their function improved.
Obviously, if you applied these drugsinto a cell that was dividing, you
would have the opposite effect becauseit would not allow it to divide.
And that's a good thing incancer where you want to stop

(12:58):
this continuous cell division.
We're excited about this.
I think it's a proofof principle approach.
Obviously, turning this into a therapeuticwould have many clinical challenges
that we would need to overcome.
But what we're doing right now iswe're focusing on alternative ways
to target the microtubules, perhapsusing other classes of drugs.

A few months later, you published again in Science Advances.
And this was looking at aparticular gene: the C9ORF72 gene.
Now this is the largestgenetic cause of ALS.
This was a study that couldpossibly impact even more people.
So tell me, what was the leadingquestion behind this study?

So C9ORF72 was discovered a few years ago.
It was a phenomenal genetic breakthroughin ALS because as you mentioned, it's
the largest genetic cause of the disease.
So it's responsible for as much as40 percent of all familial ALS, and
it's also responsible for a goodproportion of sporadic disease,
about 5, maybe 7 percent of sporadicdisease caused by these mutations.

(14:00):
Now, it's unique relative to otherALS causal genes, because instead of
a single mutation in the DNA, thisis what we call a repeat expansion.
So the way to think about this is a shortpart of the DNA, in this case, six bases,
just get expanded in people that have ALS.

(14:21):
So instead of having 10 copies of these,people that have ALS have hundreds or
maybe thousands of copies of these.
These are known as repeat expansionmutations and they can cause a number
of other neurodegenerative diseases.
So, what we knew about this mutationat the time when we started this study
was that from this repeat expansion, amutation in the DNA, we've got these

(14:45):
irregular short proteins, we callthem dipeptides, that are formed.
And these are exclusively found inpatients that have this mutation.
We knew that these dipeptides can be toxicbecause when we express these peptides
in nerve cells, basically kill them.
So we sought out to address howthese dipeptides can become toxic.

(15:06):
And using a combination of computationalapproaches as well as empirical,
experimental methods, we determined thatone of the mechanisms by which these
dipeptide proteins cause toxicity innerve cells is because they interact
irregularly with RNA molecules.
Now, RNA molecules arethese messenger molecules.

(15:29):
And what they do is they allowDNA to turn into protein.
So DNA is found in the nucleus, turnsinto RNA, and RNA makes the proteins,
which is what cells utilize to function.
Now, these dipeptide proteins basicallybind to a lot of RNA molecules.
And using a technique known as CLIP-seq,we're able to determine the precise

(15:50):
identities of the RNA moleculesthat these toxic peptides bind.
And it turns out that these RNA moleculesbelong to a specific subclass of RNA
molecules known as ribosomal RNAs.
Once we discovered that, we thought,maybe we can use this knowledge to design
a molecule that would block this toxicity.

You actually created something that you
call RNA bait in this study.
And I want you to tell me aboutthat and how you used that.

So this is something that, again, we're very
excited about, and it's inspiredby other similar discoveries
around neurodegenerative diseases.
But the simple idea is if we know whata toxic protein binds to, maybe we can
create something that looks like thingthat the toxic protein binds to in the
cell and coax the toxic protein to bindto the thing that we put in the cell.

(16:39):
We call this the bait molecule.
Because now we had uncovered that thistoxic C9ORF72 peptides bind to RNA
molecules of ribosomal identities,we designed a molecule that looked
like ribosomal RNA, but it hadchemical modifications that allowed
it to survive in cells for longer.

(17:00):
And we asked, well, if we now load nervecells from patients that have these
mutations with these RNA baits, wouldit block the toxicity of the dipeptides?
And again, to our surprise, we foundthat indeed this bait was able to
coax these proteins away from thethings that it shouldn't bind.

(17:21):
And by this fashion, wemade them less toxic.
Initially we did this in simplisticcellular models, but eventually we're
able to show that this bait moleculescan be therapeutically meaningful in
the context of human nerve cellsthat are derived from ALS patients.
So we made nerve cells that havethese mutations from patients that

(17:43):
were recruited to ALS clinics.
And when we treated these nervecells with our bait molecules,
we allowed them to survive for alot longer in our cell culture.
We then subsequently tested the abilityof these bait molecules to block the
toxicity in animal models of the disease.
And again, we found that theywere able to block this toxicity.

Finally, I want to dive into a third study.
You recently published in CellReports where you were able to use
patient derived ipsc spinal motorneuron cells to develop a model.
Tell me more about this discovery.

In this particular study, we focused on yet
another, rare genetic subtype of ALScaused by mutations in a gene called SOD1.
As we previously discussed,SOD1 was the first gene that
was ever discovered in ALS.
A lot of people have been working onthis gene for, you know, 25 years.
Now we actually, for the first time,we have a rational therapeutic approach

(18:41):
for patients that have SOD1 mutations.
And that therapeutic approach is based onthe discovery, the idea that the mutations
in SOD1 cause a disease, but what werefer to as gain of function effects.
So the protein, because it has thatmutation, it's basically doing things
that it shouldn't do in nerve cells.

(19:02):
It's becoming toxic.
And the therapeutic approach that peopleare using is these molecules known
as antisense oligonucleotides thattarget this mutant SOD1 and degrade it.
Now, while we're very excited by thefact that we have a drug that targets
a mutation and people that are treatedwith this drug seem to be doing better

(19:23):
with it, we still don't quite understandhow this mutant SOD1 protein is toxic.
How does it kill nerve cells?
And to address that question, we mademotor neurons, nerve cells from patients
that have these SOD1 mutations, andwe asked whether other proteins are
affected in their stability and functionwithin the patient's nerve cells.

(19:47):
And what we uncovered, which waspreviously unknown, was that in these
SOD1 ALS cases, one of the proteinsthat becomes unstable is a protein
known as VCP, which itself causesanother subtype of rare genetic ALS.
So again, we've got another examplewhere two distinct genetic subtypes of

(20:10):
ALS converge mechanistically in waysthat we have not previously appreciated.
So in all three studies that we'vediscussed today, the message that's coming
out is that while the genetic cause ineach one of the three cases is different,
there's always some sort of convergence,either direct: two genes that are mutated,

interacting with each other; or indirect: two distinct genes that are mutated, (20:32):
undefined
affecting the same cellular pathway.
And going back to how we started thisdiscussion, this is suggesting to us that
although the underlying cause of ALS ineach one of these three cases is distinct,
we're talking really about similarprocesses that disrupt the function

(20:56):
of the motor nerves in ALS patients.
That is important because, again, thatwill help us design, and we already have
designed, therapeutic approaches thatcould be applied to one or the other.

And I know we're talking about ALS today, but
could these approaches also helpother neurodegenerative diseases?

When it comes to our particular discoveries, they
have potential applications for othersubtypes, for other closely related
neurodegenerative disease to ALS.
The first that comes to mind isfrontal temporal dementia, which is
a type of dementia that shares bothclinical and genetic overlap with ALS.

(21:39):
So in other words, genes that cause ALScan also cause this type of dementia.
And people that have ALS can havethis type of dementia and vice versa.
A prominent example of this isthis C9ORF72 mutation that is not
only the leading genetic cause ofALS, but also the leading genetic
cause of frontal temporal dementia.

(22:02):
And in a separate project in my lab,we're trying to understand how mutation
in the same gene can cause dementia inone patient, ALS in another patient,
or ALS and dementia in a third patient.
And again, the way we're doing thatis we're making iPSC models, induced
pluripotent stem cell models, personalizedmodels from classes of patients that

(22:24):
belong to either ALS or FTD or ALS FTD.

If you could look ahead 10 years or 15 years in
the future, what would you liketo see happening in your lab?
Where would you like this to progress?

I just came back from an international ALS meeting.
It's called the InternationalMotor Neuron Disease Meeting,
which was in Basel, Switzerland.
And I witnessed what I think is amomentous event in the fight against
ALS because for the first time wewere presented with a new drug.
Again, it's an antisenseoligonucleotide drug that targets

(22:59):
an exceptionally rare type of ALS.
it usually affects teenagers.
And it's exceptionally aggressive.
And at this conference, the team ofclinicians and scientists invited to
the ceremony an actual ALS patientthat had this disease, who was
able to walk up and down the stage.
And what's incredibly powerful isbefore this teenage girl started

(23:22):
the treatment, she was bedridden.
But once the team infused thepatient with this particular
therapeutic, they were not onlyable to halt the disease progression,
but make the patient feel better.
It was a very powerful moment.
You know, there was a standingovation for both the team of
scientists and the patient.
And when you ask me what Iwant to see over the next 10
years is more events like that.

(23:43):
We're at a critical time point where,we've got this coming together of
incredible resources, incredibletechnologies the coming together
of the community to make meaningfultherapeutics for these patients
that have been really helpless.

I'm sure for a lot of people listening that gives
them a lot of hope too for folkswho have loved ones or who are
interested in the science progressing.
So thank you so much for sharing thatstory and talking me through these
recent discoveries, and I hope thatwe can have you back on in the future
to see where things have progressed.

Well, thank you so much for having me.
It was a real pleasure chatting with you.
I'd love to come back again.

Thanks for listening and be sure to subscribe to this show
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Also for medical professionals,this episode of Breakthroughs
is available for CME credit.
Go to our website, feinberg.
northwestern.
edu and search CME.

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