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Seminar Notes from October 26, 2006

Amy’s Introduction
Goals:  Bring researchers together to pool knowledge, identify gaps, identify researchers, and bring them together - to brainstorm a treatment for LAM and TSC.
Sabatini
Topics

• Talk about new data
• Challenging questions – is rapamycin and the pathway the answer/solution? If not, what is?

Slide 1 – mTOR plays a key role in growth control and various diseases such as, diabetes, LAM/TSC, and cancer.  It is implicated in growth of cells, organisms, organs.  mTOR has fundamental roles in growth control.
Slide 2 – mTOR doesn’t act alone. Acts with raptor and GβL.  How is mTOR regulated?  What is upstream?  TSC1 and TSC2 major upstream regulators of mTOR.  Finding also connects it to other diseasease
Slide 3 – connection between PI3K and TSC1, tho controversial.  Inhibitory effect downstream - mTOR inhibits pi3 kinase.
Slide 4 – random event, study of rapamycin led to discovery of mTOR pathway.  Rapamycin is immunosuppressive, but studied for sporadic tumor effects, cardiology (bad press with side effects)
Slide 5 – identified complex.  Became clear that mTORC2 existed as well.  Shares some components (rictor not raptor).  mTOR1 made sense.  mTOR2 as part of Akt pathway?
Slide 6 – highly criticized (role of mTOR 2 in Akt phosphorylation)
Slide 7 – motivation for work? Prove mTOR 2 regulates Akt.  Explained with characterization of mice
Slide 9- all components are encoded by essential genes
Slide 10 – mTOR null animal dies quickly.  Null mouse blastocyst does not grow.  Reason so bad? Knocks out both complexes.  Assumption? Raptor or rictor knockout (specific) less severe.  Not true.  Survives a little better, but essentially the same.  Suggests that loss of GBL looks like mTOR null.  Also false.  GBL null are fine.  When does GBL null animal die?
Slide 11 – day 10.5.  but has defects.  Rictor null and GBL null death same time.  Phenotypically highly similar.  Not sure why animals die yet – vascular defects, reduced/absent blood vessels, defects in placenta (Slide 12)
Slide 12 – knockouts of 4 components in 2 classes: mTOR1 and mTOR2 loss of function.
Slide 13 – what is function of mTOR2?  What happens to Akt phosphorylation in rictor null animals?  No Akt function.
Slide 14 – mTOR2 upstream of Akt.  Odd? GBL protein in both complexes.  When acutely knocked out GBl, inhibition of S6Kinase.  Chronic loss of protein, yet no defect in animals suggesting loss of mTOR1.  mTOR1 doesn not care about chronic loss of GBL protein.
Slide 15 – why does it not care?  mTOR1 has raptor, mTOR2 has no rictor with absence of GBL.   Immunoprecipitate raptor/rictor.  Loss of protein selectively disrupts selectivity/stability of rictor complex
Slide 16 – Akt substrates: TSC2, GSK3.  in null for rictor:  one site for Akt gone, other not affected.  Strong reduction of FOXO3, but not other components (incl GSK3).  Why selective?  High Akt activity?  Or other kinases can phosphorylate on sites as Akt?
Audience: Why does hemi-phosphorylated Akt signal to some effectors but not others? What does GβL do for mTOR1?
Sabatini:  No evidence for substitute.
Aud: What happens to Akt activity?
Sabatini: 10 fold effect.  No insulin induced increase in activity.
Aud:  Tried rapamycin… To increase mTOR1?
Sabatini:  little more binding, only around 10 – 20 percent…  in vitro phosphorylations tend to show false.  Could be the result of substituting kinases.
Loss of phosphorylation due to FOXO.   
Slide 17 – mTOR2 regulates Akt, close to publishing.  Signaling important to FOXO.
Slide 18 – trying to be provocative.  Open questions around mTOR pathway for TSC/LAM

• Is rheb the disease relevant effector of TSC1/2?  Is mTOR disease relevant target of TSC/rheb?  Relevant, but not conclusive relevance.
• Is mTOR the thing to target, or something else, i.e. S6Kinase?  Inhibiting mTOR long term may have bad effects.  Downstream for less toxicity?  Removal of inhibitory signal with rapamycin in mTOR1 will inhibit mTOR2 in long run.  Result is highly varied.  Finally, output in rapamycin treated people/animals not known. 
• Will TSC/LAM differ in regards to previous questions?

Slide 19 - Drosophila shows rheb/mtor connection.  Loss of TSC2
Slide 20 – cells treated with rapamycin get long dendritic spines.  We generally think of rapamycin and TSC acting in opp directions.  Not so.  Why is phenotype worse?  How could rapamycin reset TSC? 

Audience: Why does it lengthen if downstream? 
Sabatini: clearly complex system, some mTOR function important.  Suggests that for normal dendritic spine formation mTOR is necessary.  – mTOR and TSC balance, tricky gene removal.
Audience: since rapamycin also regulates the calcium signal, may have some contribution to function of neuron. 
Sabatini: FKBP has normal functions which can be suppressed by rapamycin, but requires high doses.  At lower doses shown wouldn’t expect such effects to be present..  
Audience: shTOR(?) experiment? Even possible?

Slide 21 – TSC signals to two things, rheb downstream – to mTOR.  A story with holes though.  (back two slides) what would be an experiment to rule out TSC as key in pathway? 

Audience: In the mouse trial, rapamycin is a wonder drug, but does it differ in humans?  One of the tumors (endothelial cells) TORC2 very sensitive to rapamycin.
Audience 2: It is necessary to distinguish mTOR and pathogenesis.  mTOR primarily controls proliferative part, obviously.  There is a difference between therapy and pathogenesis. 
Aud 3: The problem is in experiments with mice, there are only TSC2 mutations.  They are completely dependent on mTOR.  In other cancer types, there are PI3K mutations.  Other pathways lead to survival and growth. 
Sabatini : cml model?  What’s the evidence for lam? 
Aud 3: in other mutations, not gleevec alone. 
Aud 4: in aggressive lesions, there are other hits
Sabatini: with defined lesions, rapamycin makes sense, in real life other things are going on.  How do we address that? 
Aud 3:  phenotype the subject more completely.
Sabatinit: Akt as marker of Akt highly variable. Rapamycin always wipes out S6.
Aud 5: Any clinical trials?
Aud 6: No recruiting for LAM.  TSC + LAM sure.  No plans to biopsy lam lesions. 
Aud 3:  Any plans to obtain biopsy before trial?
Aud 7: NIH biopsy before trials in LAM to look for Akt. 
Sabatini: Effects of rapamycin acute and chronic. 
Aud 3: Is there feedback in S6? Are people looking?  Difficult for tissue excess. 
Sandy’s rep:  Are there any ideas for trials?  Any biomarkers in serum or urine to see if feedback loop is interrupted/intact?
Aud 3: Is there a biopsy pretrial?
Sandy’s rep: There is an MRI administered to assess size of lesion only.
Sabatinit: We expect rapamycin to be a wonder drug. 
Aud 8: The mouse model needs to mimic human disease.  Rapamycin effective in two losses (nf1(?) goes largely through TSC) 
Sabatini:  Why are some inputs so differently effected by TSC(-)? 
Aud 2: A TSC overlap/P10 loss?
Aud 3: LAM malignancy like cancer.  Terminology debate… 
Sabatini: Genetic evidence needed to show mTOR is key.  Evidence is using rapamycin as a tool.  Drug has a lot of peculiar aspects.  Therapeutically it works, but little understanding in solidifying connection.

Embryonic lethality?  Conditionals
Types of tumors scoring? Kidney. Planning on scoring rictor/raptor.
If in human trials things aren’t going well

Aud 3: problem is between TSC and mTOR, branches not hitting, parallel mTOR independent pathways.
Sabatini: too much literature that show other branches, can’t be ignored.
Aud 9: Are there knockout animals for specific mTOR?
Sabatini: a lot of FOXO.  Crosss of TSC to S6 kinase?  S6K on radar for diabetes.  Not for tumor models.  Maybe for these tumor models.
BREAK
Manning
Topics
What we know about signaling events interrupted by TSC1-2 complex.  Implications for TSC/LAM progression, treatment.

Slide 1 – TSC1-2 off.  Most of what we know revolves around activation of rheb.  It binds to other proteins, may have rheb independent functions. But in drosophila, most important target in cell growth.  What happens?  Rheb is activated, multiple downstream effectors.  In response, mTORC1 unlikely only downstream target.  Rheb also associates with b-raf, but mTORC1 only one with tumorigenesis.  1 – constituitive rheb signaline; no longer responsive to growth factors, is responsive to nutrients.  4ebp1, sk6.  function of skar?  S6K and mTORC1 in general controls ribosomal bio-genesis.  Amino acids still sensed in pathway.  Ribosome biogenesis.  Cpa dep translation important in cell proliferation.  2 – abberantly high mtoc1 signaling relative to growth factor stimulation.  (reading downstream phosphorylation, higher than in basal state). 
When lose TSC, S6K or something downstream negatively regulates P3K pathway, but nature of negative feedback loop not clear.  Loss of TSC shuts off critical cell survival pathway.  Leads to susceptibility of apoptotic… FOXO negative regulation of cell proliferation/survival.  GSK3 negatively regulates what TOR turns on.  EIF2B inhibted by GSK3.  think that negative regulation of PI3K Akt pathway important in regulation of lesion/tumor suppresion regulation.
Recent finding: S6K1 takes place of Akt, can negatively regulate ak3, important in TSC cell proliferation and LAM phenotypes.
Slide 3 – Akt, FOXO, GSK, not longer responsive to growth factors.  Liver hemangioma.  Akt signaling is off in these lesions GSK3 phosphorylation is present.
Slide 4 –
Slide 5 – similar substrates specificity in S6K and GSK3
Slide 7 – S6K1/ S6K2 not functionally redundant.  The two may be more relevant kinases in context.
Slide 8 – GSK activated with removal of growth factor.  Implies: GSK3 activity lower in serum starved TSC cells; hypothesis: Rapamycin activates GSK3 in TSC cells.
Slide 9 -   Data shows reduced activation of GSK3. 
Slide 10 – TSC2 proliferate in absence of serum – that is, mTOR dependent, rapamycin sensitive proliferation.  Treat them with rapamycin, look like wildtype.  Treat cells with rapamycin, decreases proliferation. Treatment with GSK inhibitors partially/significantly reverses the effects of rapamycin.  Knockdown GSK α and β, profound effects.  Is GSK3 inhibition significant in driving cells into cell cycle? No.  Inhibitors do not drive wildtype cells in cell cycle. Answer seems to be a combination of mTOR active and GSK3 off. 
Slide 11 – shown in reconstituted cells.  show phenotype dep of TSC2. TSC1 cells behave in similar manner.  GSK inhibitors rescue effect of rapamycin. 
Slide 12 – S6K1 and GSK3 are important in cell proliferation, as well as in controlling metabolic processes.
Slide 13 – GSK is indeed phosphoryalted in TSC.  Explains misregulation of... which leads to enhanced survival of TSC deficient neurons.
Slide 14 – In newborns with TSC lesions, myocytes are full of glycogen globules.  This observation puzzled the field.  Possible reason? GSK3 leads to increase in namesake substrate glycogen synthase activity.
Slide 15 – Specifically regarding LAM - GSK3 smooth muscle cell survival.  Hypoxia causes death in normal smooth muscle cells. Treatment with GSK3 inhibitors can save smooth muscle cells from death. 
Slide 16 – ELT3 cells isolated from uterine tissue.  TSC2(-) that are smooth muscle like, GSK3, inhibited by rapamycin.
Slide 17 – Differential regulation of Akt targets.  GSK3 does not take full place of Akt  with Akt substrates.  Very few targets…
Slide 18 – What happens to downstream factors with rapamycin treatment?  What to expect? Critical translation target of pathway HIF1α elevated in TSC knockout context.  Controls many (metabolic) genes, controls production of VEGF (when secreted drives angiogenesis).  TSC tumor needs to be looked at as tumor and things secreted by tumor. Predictions?  mTOR1 inhibitors relieve negative regulation PI3K pathway.  In context of fully formed tumor?  Rapamycin increases receptiveness to serum.  Little effect on serum starved cells.  Some activation in some settings (basal Akt). With rapamycin and present growth factors, implicated cell survival (possibly).
Slide 19 – rapamycin (and analogues) enhance survival of TSC(-) cells.  Feed TSC knockout larvae, can rescue them on to further stage in development.  Realistically, TSC patients are not homozygotes(?), only tumors.  Rapamycin increases cell survival, IGF1 responsiveness and IGF1 induced survival.  Rapamycin rescues TSC(-) cells from DNA damage induced cell death.
Slide 20 – Rapamycin may eliminate ability of tumor to apoptose.  In 6 patients: striking effect of rapamycin after 2.5 months on, 4 months off (significant effect of growth) indicates that cells within tumor are still present but smaller with rapamycin.  Lesions in TSC/LAM may behave differetently (due to differing vascularity).  Indicates chronic use of rapamcyin to achieve desired effects, although such high doses are not suggested (toxicity/side effects).
Slide 21 – rapamycin inhibits TORC1 in endothelial cells.
Slide 22 – Drive Akt activation in endothelial cells get path angiogenesis.  Rapamycin inhibts phosphorylation in endothelial cells, and activates Akt phosphorylation in tumor cells.
Slide 23 – Implications: rapamycin inhibits TORC2 in angiogenesis. May be more useful in understanding… what happens to TORC2 in TSC lesions?  Why does TORC2 respond in some cells and not others?
Slide 24 – Rapamycin activates Akt in human carcinomas. 
Slide 25 – elts cells mimic LAM.  Rapamycin activates Akt in TSC2(-) cells. 
Slide 26 – Data suggests perhaps rapamycin will be cure all.  However, we don’t know what’s going on in the tumor in regards to pathway.  What if: Rkt inhibitors, PI3K inhibitors, Akt inhibitors along with rapamycin?  Best inhibitor would be mTOR kinase domain inhibitor (inhibits both mTORC1/2.  Important: questions of toxicity).

Audience: Important to know status of PI3K pathway in patients on trial
Manning:  Even if Akt is up, may not make tumors worse, but will not be a cure.  But may just be a correlation (indicating resistance to drug)
Aud:- elt study (?) 
Manning: (references) current studies
Aud: P10 loss, combination of mTOR /PI3 inhibitors look attractive, but…
Manning:  most combinations Akt(-) is lethal. 
Aud: EIF2B.  looked at activity? Effect on translation?
Manning: Haven’t looked at differences.  GSK3 inhibitors do not affect rapamycin ability of mTOR inhibition.
Aud: why advantage for tumor to store glycogen?
Manning: maybe glycogen synthesis is turned up?  Non-muscle cells don’t stroe glycogen.  Smooth muscle cells may
            Lam cells may.
Advantage or offshoot?  GSK3 negatively regulates glycogen synthesis. 

Aud:  in patients with TSC, hormonal regulation with shrinkage in relation to TSC?
Manning: don’t know if they are related.  Most interesting that tumors go away.  Small spike later on during puberty.  GSK3 inhibition may contribute to that one property of tumor.
Aud:  Models in studies?
Manning: Some patients on lithium.  Rapamycin may be reversed by lithium.  GSK3 has many targets.  Most of its targets are proteins involved in G1S transitions.  If you could activate GSK3, possibly worth looking into.  (inhibit Akt).
Aud:--  
Manning: location, speculation of loc combined with threshold of activity that Akt is relevant kinase.  Don’t know what causes substrate specificity.