Finding New Treatments By Understanding Meningioma Biology

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00:00:08:24 I'll be honest, I'm lazy.
00:00:11:10 And so are my cells.
00:00:13:11 Actually, yours are too.
00:00:16:00 Turns out each of our cells have 3 billion base pairs of DNA
00:00:22:03 and they simply don't wanna go through all of that.
00:00:25:17 So instead, they place bookmarks to remind themselves
00:00:29:12 which parts are or are not important.
00:00:32:29 These bookmarks are chemical modifications
00:00:35:20 to the DNA itself.
00:00:37:23 I study one of those modifications known as DNA methylation.
00:00:44:06 And I try to see its relevance
00:00:46:16 in the type of tumor known as meningioma.
00:00:50:08 My central research question is
00:00:52:28 can we use DNA methylation to understand meningioma biology?
00:00:59:03 But before we can get into that,
00:01:00:22 we have to understand what meningiomas are
00:01:03:13 and what DNA methylation is.
00:01:06:10 Meningiomas are the most common intracranial tumor,
00:01:10:25 intracranial simply means that they're inside of our skull.
00:01:14:28 This is an MRI of a patient with a meningioma
00:01:18:27 at the top of the screen is the patient's eyes.
00:01:21:29 And at the bottom of the screen
00:01:23:08 is the back of the patient's head.
00:01:25:23 Near the back of the patient's head,
00:01:27:20 you can see a slightly different colored sphere,
00:01:30:26 that's the meningioma.
00:01:32:27 Meningiomas grow from the meninges
00:01:35:11 which is the layer of tissue
00:01:37:03 in between our brains and our skulls.
00:01:40:16 Currently, the only treatments available for meningioma
00:01:44:12 are surgery and radiation.
00:01:47:10 These treatments work well for many patients
00:01:49:24 but there are two major problems.
00:01:52:26 First of all, these treatments have side effects.
00:01:56:07 As you can imagine,
00:01:57:15 getting brain surgery or exposing your brain
00:02:00:05 to radiation are not trivial operations.
00:02:04:08 Second of all, some meningiomas come back
00:02:08:20 even after being treated with the risky techniques
00:02:11:27 again and again.
00:02:14:01 And at that point there simply aren't any more options left.
00:02:18:12 There aren't any medications to treatment meningioma
00:02:21:18 and that's because we don't understand the biology
00:02:24:21 of meningioma.
00:02:26:07 We don't know what pathways or genes
00:02:30:02 to target with medications.
00:02:32:23 So that brings me back to my central research question.
00:02:37:19 Can we use DNA methylation to understand meningioma biology?
00:02:44:08 So what's DNA methylation,
00:02:46:14 and why would we wanna use that to understand meningiomas?
00:02:51:07 Well, DNA makes up our genome
00:02:54:10 and our genome is like a very, very long book
00:02:57:25 with 3 billion letters.
00:03:00:12 But instead of the 26 letters of the alphabet
00:03:03:25 it has four letters, four base pairs.
00:03:07:20 These are adenine thymine, guanine, and cytosine.
00:03:14:00 Sometimes cells add a methyl group two cytosines,
00:03:18:00 this is a chemical modification to the base pair.
00:03:22:05 This is what DNA methylation is.
00:03:25:13 These methyl groups act as bookmarks to tell the cells
00:03:28:21 which parts are or are not important.
00:03:31:28 Now, if you or I read the same book
00:03:35:27 we might highlight different passages
00:03:38:01 or place bookmarks on different pages.
00:03:41:01 I might think pages one, three, and five are important
00:03:44:12 while you might think pages two, four and six are important.
00:03:48:29 Similarly different cells, place DNA methylation marks
00:03:53:14 in different parts of the genome,
00:03:55:08 depending on what they find important.
00:03:58:03 For instance, your brain cells will have different patterns
00:04:02:09 of DNA methylation compared to your heart cells.
00:04:07:08 Similarly different types of tumors
00:04:10:04 also have different patterns of methylation.
00:04:13:03 In fact, one study found that DNA methylation patterns
00:04:17:21 are the best way to distinguish and understand the biology
00:04:22:04 of central nervous system tumors, which include meningiomas.
00:04:27:25 And we can actually get more granular than that.
00:04:31:04 As you may recall, there are different kinds of meningiomas.
00:04:34:13 Some are aggressive
00:04:35:18 and come back even after being treated again and again.
00:04:40:02 We can use DNA methylation patterns
00:04:42:21 to understand the differences
00:04:44:16 between different kinds of meningiomas.
00:04:48:03 So how do we study DNA methylation?
00:04:51:25 Well, tumors are made up of a bunch of cells.
00:04:55:28 So the first step is to extract the DNA from those cells.
00:05:00:08 Then we place the DNA on a special chip.
00:05:03:21 What this chip does, is look simultaneously
00:05:06:29 at 850,000 cytosines across the genome.
00:05:12:14 And it calculates for each of those locations
00:05:16:13 the percentage of DNA that is, or is not methylated.
00:05:21:21 Then we can take that data and look for patterns
00:05:24:22 in where those methylation marks are.
00:05:28:24 In order to find patterns
00:05:30:06 in the DNA methylation of meningiomas,
00:05:33:22 we had together a lot of meningiomas
00:05:36:08 and ideally from more than one place.
00:05:38:27 We need a lot to make sure that the patterns we find
00:05:41:22 are real.
00:05:42:28 And we wanted just look at meningiomas
00:05:45:14 from more than one location
00:05:47:07 to make sure that the patterns we find
00:05:49:15 are true for all meningiomas
00:05:51:14 and aren't specific to a particular geographic area.
00:05:55:20 So in order to study the DNA methylation of meningiomas
00:06:00:02 I gathered 565 meningiomas.
00:06:04:06 200 from UC San Francisco
00:06:07:07 and another 365 from our collaborators
00:06:10:20 at Hong Kong University.
00:06:12:28 And I started looking at their methylation patterns.
00:06:16:14 This is what I found.
00:06:18:10 Now, I know this is a huge mess,
00:06:20:21 but let me walk you through it.
00:06:22:25 This is what's known as a heat map.
00:06:24:28 And the first thing you need to know
00:06:26:22 is that red means that there's more methylation
00:06:29:12 and blue means that there's less methylation.
00:06:33:15 Each of these rows is a different cytosine,
00:06:36:10 a different location in the genome that is
00:06:39:04 or is not methylated.
00:06:41:24 Each of these columns is a different tumor,
00:06:44:12 a different meningioma.
00:06:46:27 Now you're probably still having difficulty making sense
00:06:49:10 of this heat map.
00:06:50:20 And that's okay,
00:06:52:02 because we can't just look at these heat maps
00:06:54:14 and figure out what the patterns are.
00:06:56:14 We actually have to use computer algorithms
00:06:59:14 to group the cytosines
00:07:01:11 and tumors together to try and find patterns.
00:07:04:29 So that's exactly what I did.
00:07:06:27 I used a couple of computer algorithms to look for patterns
00:07:10:03 in this data.
00:07:11:13 And this is what I found.
00:07:13:09 That meningiomas are comprised of three subgroups,
00:07:16:19 which I'm calling A, B and C.
00:07:19:24 And I found that this was true, both in the meningiomas
00:07:23:03 from UCSF, as well as the ones from Hong Kong University.
00:07:28:03 Now, just because an algorithm tells us
00:07:30:15 that there are different groups of meningiomas
00:07:33:10 doesn't necessarily mean that those groups are meaningful
00:07:37:05 for patients or for the biology of meningioma.
00:07:41:10 So to see if these groups were clinically relevant
00:07:44:24 I decided to look at their clinical outcomes
00:07:48:14 and to do that I looked at something known as
00:07:50:25 absence of recurrence, which we're gonna see
00:07:53:08 on the Y-axis of this graph.
00:07:55:28 That's basically the percentage of meningiomas
00:07:58:29 that have not recurred or comeback, after being removed.
00:08:03:23 On the X-axis we have time with the time zero being
00:08:07:21 when the meningioma was initially removed with surgery.
00:08:12:05 Now, what you're gonna see is that all the curves
00:08:14:10 are gonna slope downward.
00:08:16:05 And that's because as time goes on
00:08:18:20 meningiomas are more and more likely to come back.
00:08:23:04 But what we see is that these three sub groups
00:08:25:21 of meningiomas slope downward at different rates.
00:08:30:03 What that tells us is that these three sub groups
00:08:32:25 have different outcomes.
00:08:34:27 Some of them recur faster than others.
00:08:37:29 Today in particular,
00:08:39:11 I'm interested in subgroup C meningiomas the ones in red.
00:08:43:25 And that's because they slopped down the fastest
00:08:46:16 meaning that those are the most aggressive tumors
00:08:49:12 that come back even after being treated again and again.
00:08:54:26 So what makes subgroup C meningiomas different?
00:09:00:01 We found a couple of different things.
00:09:02:07 First, we looked at CNVs or copy number variations.
00:09:07:23 Sometimes when tumor cells are growing and dividing
00:09:11:02 they make either too few or too many copies of their DNA.
00:09:16:17 And certain pieces of the genome might have too many
00:09:20:08 or too few copies as a result.
00:09:22:29 We can actually quantify what percentage of the genome
00:09:26:02 have these variations.
00:09:28:11 And what I found is that subgroup C meningiomas
00:09:31:26 had the most copy number of variation
00:09:34:06 compared to the other two subgroups.
00:09:36:29 The other thing we found unique
00:09:38:16 about subgroup C meningiomas,
00:09:40:18 was that they had an increased proliferation.
00:09:43:23 Proliferation is the percentage of cells in a tumor
00:09:46:28 that are actively dividing.
00:09:49:02 The more cells that are dividing,
00:09:50:25 the more quickly these tumors grow.
00:09:54:03 Now as a cancer biologist,
00:09:56:00 when I see an increase in proliferation
00:09:58:13 I start thinking about the cell cycle.
00:10:01:22 The cell cycle is a series of steps that cells take to grow
00:10:05:20 from one cell into two cells.
00:10:09:00 And then those two cells can take the same steps
00:10:11:18 and become four cells and so on and so forth.
00:10:16:08 These steps are driven by proteins
00:10:18:20 known as cyclin-dependent kinases or CDKs.
00:10:24:01 Each CDK and each step of the cell cycle
00:10:27:10 is regulated tightly by other proteins
00:10:30:04 that act as traffic signals that tell the CDKs when they can
00:10:34:05 or cannot go to the following step .
00:10:37:06 In normal cells this entire process work seamlessly.
00:10:41:05 However, in cancer this process is often dysregulated
00:10:45:04 and cells and tumor cells can grow uncontrollably.
00:10:50:18 In fact, in some instances, copy number variation
00:10:54:24 which if you remember is one of the things we saw
00:10:57:09 in subgroup C meningiomas, has been linked
00:11:00:19 to cell cycle dysregulation.
00:11:03:24 In particular, a copy number loss
00:11:06:25 of a particular piece of DNA known as CDKN2A/B.
00:11:12:28 The genes on this piece of DNA create two proteins
00:11:17:02 known as P16INK4A and P15INK4B.
00:11:22:17 These proteins act as stop signs
00:11:25:03 for two CDK proteins known as CDK4 and CDK6.
00:11:31:00 As a result, in some tumors when this piece of DNA is lost
00:11:35:05 because there are too few copies
00:11:36:28 the proteins aren't made anymore.
00:11:39:00 As a result, the cells can grow
00:11:41:05 and divide because there's nothing stopping the CDK4
00:11:44:28 and CDK6 proteins.
00:11:47:22 Now, this has been seen in many other cancers
00:11:50:01 but is this relevant in meningioma?
00:11:53:22 What we found is that meningiomas
00:11:55:27 with loss of this piece of DNA
00:11:58:04 had far worse at clinical outcomes
00:12:00:20 compared to meningiomas without loss.
00:12:03:16 In addition, when we looked at meningiomas with loss
00:12:07:04 of this piece of DNA, a majority of them
00:12:09:26 were subgroup C meningiomas.
00:12:12:19 This made us confident that loss of CDKN2A/B
00:12:17:02 is one of the ways in which subgroup C meningiomas grow
00:12:21:09 and divide.
00:12:22:28 However, this is it, the whole story.
00:12:25:19 We also noticed that about a quarter of meningiomas
00:12:29:09 that don't have loss of CDKN2A/B
00:12:32:12 are still subgroup C meningiomas.
00:12:34:24 What this tells us is that subgroup C meningiomas
00:12:38:25 must also have other ways to increase their proliferation,
00:12:42:25 to grow and divide.
00:12:45:13 So we wanted to see
00:12:47:11 what else might be regulating CDK4 and CDK6.
00:12:51:24 And in order to do that, we decided to look at something
00:12:54:22 known as histone acetylation.
00:12:57:29 Histones are proteins around which DNA is wrapped.
00:13:02:01 Sometimes these histones have chemical modifications
00:13:05:15 known as acetylation attached to them.
00:13:08:27 DNA is the first step in an assembly line
00:13:12:12 or a factory that makes the proteins such as CDK4/6.
00:13:17:24 And histone acetylation acts as
00:13:20:23 kind of like a neon open sign that tells us
00:13:23:06 that the DNA is open and ready to create protein.
00:13:28:18 And so, as a result we want to look at
00:13:30:23 acetylation across the genome.
00:13:32:28 And in order to do this, we used a technique
00:13:35:16 called histone acetylation chip sequencing.
00:13:39:07 And in particular,
00:13:40:13 when we zoomed in on CDK6 in particular,
00:13:44:26 we found that the gene CDK6 had quite a bit
00:13:48:10 of acetylation signal, as you can see in red.
00:13:52:15 Of note we saw a slight dip in the signal near the peak.
00:13:56:16 And what that told us is that there was someone
00:13:59:07 or something in front of the open side,
00:14:02:10 blocking it, making the signal a little bit less.
00:14:06:28 However, based off of where exactly that dip is
00:14:10:20 and the shadows cast by it
00:14:12:27 as represented by these gray boxes,
00:14:15:27 we can actually figure out what it is.
00:14:18:13 And what we found is that it's a protein called USF1.
00:14:23:16 USF1 is a transcription factor.
00:14:25:29 You can think of it kind of like a factory foreman
00:14:28:22 that can turn genes on or off
00:14:31:08 therefore controlling the amount of protein made.
00:14:35:11 USF1 of note is on chromosome 1Q
00:14:39:11 which is often gained in meningiomas meaning that
00:14:42:17 some meningiomas have more USF1 than normal.
00:14:46:14 In fact, when we look at meningiomas with the gain of USF1,
00:14:50:26 we see that a vast majority of them
00:14:52:28 are sub group C meningiomas.
00:14:55:26 In addition, when we look at the clinical outcomes
00:14:58:22 of patients with meningiomas with the USF1 gain,
00:15:02:16 we see that they have far worse clinical outcomes
00:15:05:13 than patients with meningiomas without USF1 gain.
00:15:09:24 Now, just because USF1 is at the CDK6 gene
00:15:14:11 doesn't necessarily mean that it's turning CDK6 on or off.
00:15:19:22 So in order to know that for sure, we decided to decrease
00:15:23:11 and increase the amount of USF1 in meningioma cells.
00:15:27:27 And this is what we found.
00:15:29:24 When we decrease the amount of USF1,
00:15:32:11 we saw a decrease in the expression of CDK6.
00:15:36:06 When we increase the amount of USF1,
00:15:38:19 we saw an increase in the amount of CDK6.
00:15:42:16 This made us confident that USF1
00:15:45:01 was indeed controlling the CDK6 and turning it on and off.
00:15:50:22 So to recap, subgroups C meningiomas seem to grow
00:15:55:03 and divide through the CDK4/6 proteins.
00:15:59:21 And they do this in a couple of different ways.
00:16:02:00 They either get rid of stop signs,
00:16:03:21 such as the P16 and P15 proteins
00:16:07:08 or they use USF1 to turn on CDK6 even more.
00:16:12:17 Now, this is really interesting
00:16:14:02 about the biology of meningioma
00:16:16:09 but what does this tell us about meningioma patients?
00:16:20:00 Does this change anything for them?
00:16:22:16 Well, it turns out there are some drugs
00:16:24:13 known as CDK4/6 inhibitors.
00:16:27:17 That act as really big stop signs to stop CDK4
00:16:31:08 and CDK6 from going to the next step in the cell cycle.
00:16:35:28 These drugs are actually already FDA approved
00:16:38:25 to treat certain kinds of breast cancer
00:16:41:18 but they've never been used to treat meningioma.
00:16:45:13 We wanted to see whether or not these drugs
00:16:47:17 could block meningioma growth.
00:16:50:00 In order to do that,
00:16:51:10 we used a technique known as clonogenic growth assays.
00:16:55:27 What we do is that we grow meningioma cells
00:16:59:03 on a piece of plastic,
00:17:00:25 and then we stain the cells purple
00:17:03:01 and we can see how many cells grew.
00:17:06:01 We can also treat the cells with drug while they're growing.
00:17:09:10 And ideally, we wanna see something like this.
00:17:12:14 Far fewer cells when the, when they're treated with drug
00:17:15:27 compared to when they're not.
00:17:17:23 And we can actually quantify
00:17:19:08 what the percentage of cells is in the drug treatment
00:17:22:20 compared to when there isn't drug treatment.
00:17:25:14 And that's what we're gonna see on this graph on the Y-axis
00:17:28:19 the percentage of cells compared to no drug treatment.
00:17:32:15 And on the X-axis,
00:17:33:22 we're gonna see different doses of drug.
00:17:36:16 And in particular,
00:17:37:17 we actually use three different CDK4/6 inhibitor drugs.
00:17:42:11 And what we found is that all of them seem to block
00:17:45:10 the growth of meningiomas.
00:17:47:20 I should note that these meningioma cells that we used
00:17:51:04 have the same methylation patterns
00:17:53:08 as subgroups C meningiomas.
00:17:56:09 In addition, we also repeated this
00:17:58:10 with two other types of meningiomas cells
00:18:00:29 that also have those methylation patterns
00:18:03:11 and the drugs worked in all of them.
00:18:06:07 This made us feel confident
00:18:08:06 that these drugs might be a viable strategy
00:18:11:01 to treatment meningioma.
00:18:13:02 But, simply growing cells on plastic
00:18:16:03 isn't the most realistic way to study meningioma.
00:18:19:15 As a result, our lab has developed a new methodology
00:18:23:07 to look at meningioma called organoid co-culture.
00:18:27:21 This system has two parts.
00:18:30:14 First on the inside of the dotted white circle in green
00:18:34:29 are normal brain cells known as astrocytes
00:18:38:16 and they form this large sphere known as an organoid.
00:18:42:12 You can think of it kind of like a mini brain.
00:18:45:14 On the outside of the dotted white circle in red,
00:18:48:21 we have meningioma cells.
00:18:51:07 Now watch what happens when we let these cells grow together
00:18:55:08 in something known as 3D culture.
00:19:00:18 As you can see the meningiomas form smaller spheres
00:19:04:02 on the outside of the organoid
00:19:06:11 and we call these tumorspheres.
00:19:08:26 And this is actually very similar
00:19:10:16 to how actual meningiomas forms spheres
00:19:13:11 on the outside of patients' brains.
00:19:16:06 As a result, we think that this is a much more
00:19:19:04 realistic way to study meningiomas
00:19:21:17 because it mimics how actual meningiomas grow.
00:19:25:26 So we decide to use the system
00:19:28:08 and treat these meningioma cells
00:19:30:10 with Abemaciclib one of the CDK4/6 inhibitor drugs.
00:19:35:20 And this is what we found.
00:19:38:15 What we see is that treating meningioma cells
00:19:41:26 with Abemaciclib decreases the number of meningioma cells
00:19:45:23 on the outside of the organoid.
00:19:48:09 A low dose of Abemaciclib decreases it a little
00:19:51:16 while a high dose decreases it more.
00:19:54:28 This made us feel even more confident
00:19:57:09 that these drugs would be able to help patients
00:20:00:10 because this is a much more realistic system.
00:20:03:28 We're also using other systems to study this as well.
00:20:07:03 We've injected mice with meningioma cells
00:20:09:26 and are treating those mice with these drugs.
00:20:12:16 And the initial results are very promising.
00:20:15:18 However, we realized something while doing all of this
00:20:19:17 these drugs are already FDA approved.
00:20:22:28 That means that we can actually start helping patients now.
00:20:27:08 So we've started working with some of the physicians at UCSF
00:20:32:08 to use these drugs on an experimental basis
00:20:36:00 to treat patients for whom all other treatments have failed.
00:20:40:09 And I just wanna walk through
00:20:41:26 the treatment course of one of those patients
00:20:44:23 to show what a difference this has made.
00:20:48:03 On the Y-axis here, we have meningioma volume
00:20:51:23 or how large the tumor is.
00:20:54:04 On the X-axis, we have time.
00:20:56:17 With times zero being when the tumor was first diagnosed.
00:21:02:12 What we see is that the patient initially received surgery
00:21:05:25 which removed a majority of the tumor, but not all of it.
00:21:09:28 Then like in the case of many aggressive meningiomas
00:21:13:13 the tumor started growing again.
00:21:16:01 She received radiation, but it kept growing.
00:21:18:27 She received a different experimental therapy
00:21:21:26 but that didn't help.
00:21:23:14 She even received a second round of radiation
00:21:26:04 but the tumor continued to grow.
00:21:29:00 It was at that point
00:21:30:02 that she was started on Abemaciclib.
00:21:33:03 And what we see is that for the first time,
00:21:35:28 since her initial surgery, the tumor started shrinking
00:21:40:10 and perhaps more importantly,
00:21:42:28 some of her symptoms started to improve.
00:21:46:09 Now, this one patient doesn't prove
00:21:49:11 that this drug is a cure.
00:21:51:09 And there are still many questions
00:21:53:03 that remain to be answered
00:21:54:21 such as which patients will benefit the most
00:21:57:19 from these drugs.
00:21:59:01 But this work sets the stage for future clinical trials
00:22:03:12 to answer exactly those questions.
00:22:07:29 As I finish this talk,
00:22:09:17 I wanna remind you of this one thing.
00:22:12:13 All of this work started
00:22:14:06 by looking at patterns of DNA methylation.
00:22:17:21 Without that we wouldn't have found subgroups C meningiomas
00:22:21:26 and without studying subgroups C meningiomas,
00:22:24:20 we wouldn't have identified CDK4/6 as a target.
00:22:28:29 Only by understanding the biology of meningioma
00:22:32:12 were we able to find a potential new treatment.
00:22:36:26 Thank you.

This Talk

Speaker: Abrar Choudhury

Audience:

Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: November 2020

Talk Overview

Meningioma is the most common intracranial tumor, with limited treatment options. In order to identify new therapies for meningioma, it is important to understand their underlying biology. In this Share Your Research talk, Dr. Abrar Choudhury describes his thesis research, which began by looking at DNA methylation patterns to better understand gene regulation in meningioma, leading him to identify the cell cycle proteins CDK4 and CDK6 as therapeutic targets.

Speaker Bio

Abrar Choudhury

Abrar Choudhury completed his PhD in Biomedical Sciences at UCSF, and will receive his MD from UCSF in 2023. He studies the underlying biology of brain tumors with the goal of identifying novel therapeutic targets. https://twitter.com/abrarchttps://raleighlab.ucsf.edu/raleigh-laboratory Continue Reading

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Brittany Anderton (iBiology): Producer
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