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- Thank you very much for inviting me to speak here.
I'm going to be talking
about targeting stem cell signals
in therapy resistant cancers
in solid and heme malignancies.
And I'll just start sort of generally with the idea
that we have for many years now been interested
in how pre-malignant lesions
with additional mutations become malignant.
And one of the things that we've worked
on is not as much proliferation or cell death,
which are actually apparent in both states,
but the fact that there's a state change that occurs
when pre-malignant lesions become malignant,
and they undergo differentiation arrest
and are unable to differentiate further.
So if you think about development, where normally you go
from being immature to being mature.
In this case, it's as if these mutations are really
unraveling all the programs that were put
in place during development
so that the cells become more immature collectively.
So you can think of this sort of in development in reverse.
So in thinking of this as development in reverse,
one of the things that we've done in context
of trying to understand this is look at stem cell signals,
which as you know, are high early in development,
and they fade or are extinguished with differentiation.
And we, and many other labs, have shown that in cancer,
as pre-malignant lesions can be fairly low
in the stem cell programs, but these are elevated
or hijacked and activated
as the cancer becomes more aggressive.
So the idea of studying this really longterm is
that if we understood the signals that are turned
on to drive this pre-malignant lesion
to a full on malignancy, those may be used
as a molecular basis for enabling early detection,
and if we knew the signals that are needed
to take this pre-malignant lesion to a full on malignancy,
it may be a strategy for early interception,
and many of the pathways and drugs that
as a community we have discovered could very well
be effective or more effective early on.
And one of the challenges has been really
that we use a lot of these drugs
or interception programs too late in disease.
So in looking at stem cell programs,
one of the pathways that we've been very interested
in is an RNA binding protein called Musashi,
and this was originally identified
at Hopkins as an RNA binding protein
during Drosophila development, and when it's mutated
or there's a loss of function created, instead
of one bristle and a neuron, these flies get two bristles.
And because of its double bristled phenotype,
it was named Musashi after this iconic Japanese samurai
who used to fight with two swords.
And we had found several years ago
that Musashi was actually very low in chronic phase disease
and that it rose as disease became more
and more aggressive in chronic myeloid leukemia,
stepping through accelerated phase into blast crisis.
So we had done work showing
that if you block Musashi either through shRNA delivery
or through a genetic allele, you can gain
in survival in these models and leukemia growth,
and also the patient samples that were
in blast crisis would respond to blockade in Musashi.
So one of the things that we'd noticed
at the end of that work was that Musashi was
in fact not just high in heme malignancies,
but also in a lot of solid cancers.
And this included aggressive cancers like GPM,
high-grade breast cancer, colon cancer, and lung cancer,
but no one really knew what its role was.
So we started to work with Andy Lowy,
who's a pancreatic surgeon on pancreas cancer,
which as you know is a lethal and deadly disease
with very few options currently,
and much in need of sort of an altered clinical landscape.
So we studied this to understand
whether Musashi and other stem cell programs may be relevant
in pancreas cancer progression partly
because if you look at how pancreas cancer progresses,
it goes from being highly differentiated,
although dysplastic, into a more malignant adenocarcinoma,
which is fairly undifferentiated.
And so we took a model of pancreatic cancer called the KPC
where the RAS and P53 alleles were mutated.
And in this model, there's a very large tumor
that develops in a few weeks,
and when you delete Musashi in this,
you get a much smaller tumor.
But what the important thing is actually
it's not just a smaller tumor,
it's more benign because if you look
at a cross section of the wild type versus the knockout,
the wild type is all sheets of adenocarcinoma,
whereas the knockout still has regions
of normal tissue left, and whatever abherent tissues are
arising are in fact all PanIN,
so while the wild type has all adenocarcinoma,
the knockout has mostly PanIN lesions,
and no adenocarcinoma suggesting
that these stem cell programs are really critical
for powerful mediators like RAS and p53
to take a pre-malignant lesion into a full on malignancy.
So we've also developed reporters
to understand how these Musashi positive cells contribute
to disease, so these knocked in
so that cells are positive for Musashi are lit up
in green or yellow, so this is GFP or YFP,
and you can see that it lights up stem cells
but does not, is not active in differentiated cells,
and in the brain for example,
it lights up the hippocampus and SVZ domain,
which are enriched for stem and progenitor cells,
and it's silent in most of the rest of the mature brain.
So what we've done is crossed this to the KPC model.
Again, the pancreatic cancer model,
to really highlight the heterogeneity of the cells there.
So this shows you that you can, although everything is RAS
and P53 mutated, the GFP positive cells
and GFP negative cells are pretty clear.
And the GFP negative cells are predominantly
what the tumor is made of,
but the GFP positive cells are a small subset
of cells that carry a stem cell program.
Now, although they're a minority in reality,
they're more aggressive.
So if you take the Musashi reporter positive,
and the negative, the reporter positive makes organizer
where negative doesn't.
And the report of positive cells really drive all
of the lethality and the reporter negative cells don't.
And finally, if you treat
with standard of care chemotherapy, like gemcitabine,
what you see is that the report positive,
the stem cell part of the tumor is very small at the outset,
but as you treat with the chemo,
the reporter negative cells are depleted.
The reporter positive cells remain behind.
So they're really more resistant
to these drugs that are currently used clinically.
And they are really the main composition
of residual disease or drug resistant disease.
So this is a really potentially powerful platform
for identifying drug resistant cancers.
So this is a visual, in fact,
like a movie of a pancreatic tumors in the KPC model
highlighting the stem cells within the cancer.
A lot of the blue,
and this is imagine a large sheet,
are all RAS p53 mutated, but epigenetically,
there's a subset that's activated the stem cell program,
and you can see that it's spatially restricted.
And when you treat
and that this can be really a good model
to identify new programs and pathways that might allow us
to eliminate this population along with hemo,
which might be better at eliminating
through the bulk of the tumor.
So we've also imaged beyond solid cancers, leukemias,
and one of the things that we found in leukemias is
that this is using live imaging to noninvasive imaging
to look inside the native micro environment
to understand how cancers grow.
This is just showing you a bone marrow,
a view of the bone marrow in a living animal.
And you can see sort of the blood vessels lit up
and rest of the marrow lit up in blue and the greeners,
so the edges of the bone lit up with the reporter,
but the important thing is that when we image leukemia cells
we don't actually find them.
I mean, they are motile to some extent,
but largely also enmeshed within the marrow.
So one of the reasons we were doing this is really
to understand how much leukemia cells may depend
on the micro environment.
And what we found is that a lot
of leukemic cells are actually enmeshed and interacting
with stromal cells and cells of the microenvironment,
suggesting that they may utilize interactions
in the microenvironment as dependencies.
So the reason I mentioned those is really
because that led us looking for Musashi targets.
So we knew Musashi was important, but we were really looking
for targets that might enable cells
to interact with the micro environment.
And so we did a screen looking
for targets of Musashi in both blast crisis CML and AML,
and we looked at common genes that were downstream
of Musashi because we knew this was critical,
but we really wanted to find other downstream target
that could be actionable or could be inhibited
by new strategies.
So through this,
we identified a molecule called tetraspanin 3,
and this was downstream of Musashi
in both AML and blast crisis CML.
So large family of four pass transmembrane proteins
that can serve as a scaffolding protein,
and can associated with integrins,
allowing a sort of involvement in cell adhesion
and proliferation, but very little was really known
about it, and none really known about it in cancer.
So what we did was we inhibited the tetraspanin protein,
and showed that either through shRNA
or by building a knockout that we developed,
you could really gain in survival,
So this is survival,
this is how fast the control a mouse die,
the mice die of the tumor.
And if you inhibit tetraspanin,
there's a very clear gain in survival.
This is a mouse model.
And if you look at patient samples
what you see is if you block it either with a control
or you block it tetraspanin,
there's a very dramatic reduction in growth of the leukemia
when you block this program.
So based on this, we have launched a program really
targeting tetraspanin in context of leukemia,
and these antibodies have now been developed
with the help of CIRM and are being vetted and tested
in context of both in vitro inhibition of leukemic growth,
as well as in vivo inhibition.
And so just to summarize, what we've really found is
that interactions with the local environment can
really amplify the impact of intercellar
and oncogenic signals on leukemia stem cells
and disease propagation, and that targeting these programs
like tetraspanin 3 that we've done with the help
of CIRM may be more effective as a sort
of a combinatorial blockade of both niche signals
that support these stem cells
or these apparent aggressive stem cells within cancers,
as well as blocking intracellular events,
and collectively they may be better able to control disease.
So this is just to thank all the people who did the work.
So all of the work on Musashi and tetraspanin
and leukemia were carried out by Young Kwon
and Jeevisha Bajaj, and others involved
in the leukemia side are Kyle Spinler,
Yutaka and Mike Hamilton.
And a lot of the testing that was done
on these antibodies were done with Yutaka and Emily.
And I just like to also thank our collaborators,
Andy Lowy as well as Jeff Esko, Mark Ginsberg, Dan Von Hoff,
and also thank CIRM for their support of all of our work.
And I'd be happy to take any questions
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