I posted the first half of these questions for the researchers on this study yesterday. Here are the rest of the questions that came out of conversations after some of my friends & I broke down this research finding.
Q: Another interesting finding is that this pathway may interact with the mTOR pathway or it may not. It doesn’t even need to. It’s completely independent of that current pathway. To some of us who have only partial response from Rapamune we have always felt that there might be another pathway involved & that’s why a certain segment of our communities don’t respond favorably to our current treatment options. Did you have other theories before this study that maybe this pathway may be linked to another pathway that could also be independently functioning like this one is? Or possibly another pathway that maybe linked to mTOR but has not yet been discovered?
A: Since absence of HMGA2 leads to a complete absence of mesenchymal tumors in the TSC tumor model, we have shown that HMGA2 plays a central role and is required in tumor formation in TSC and is separate from the mTOR pathway. Our results state that in at least 50% of LAM tumors, the mTOR pathway is NOT activated. Therefore, activation of the mTOR pathway is not required or necessary for LAM tumor formation and these alternatives need to be further investigated.
Q: One of the statements that struck me personally was this one “Approximately 50 percent of the tumors do NOT exhibit altered mTOR pathway activation. Thus alternative pathways to the activation of the mTOR pathway play a major role in the pathogenesis of LAM tumorgenesis”. Could you talk a bit more about why these other tumors don’t express altered mTOR activation? Or is this just something you uncovered & are scratching the surface on to learn more as to why this occurs?
A: Our data demonstrates that it is NOT necessary for the mTOR pathway to be activated for LAM tumor formation. In fact, we hypothesize that in the 50% of tumors that activate the mTOR pathway, this activation occurs at the latter stages of tumor formation, if not after the tumor has formed. These differences could explain the heterogeneity in the patient population since the disease is at various stages in each patient.
Q: What was an interesting finding in this study was the age that the mouse models started developing tumors. Definitely not before 9 months of age. Do you have any theories as to why these tumors presented so late or at certain ages? How does this relate to how TSC & LAM both start to present in our bodies?
A: This finding indicates that other genetic events must occur within a cell, after the single hit that led to the inactivation of one TSC allele, for tumor formation to occur.
Q: Can you also address how you came to find out how this protein is present in all LAM cells & a high percentage of kidney samples? Do you have any basic theories as to how this comes to express itself in these samples?
A: Because of our background in benign mesenchymal tumors, lipomas for example, and we had identified HMGA2 as being present in these tumors, LAM was simply another example of a benign mesenchymal tumor and so we expected its presence in LAM.
Q: In discussing this finding with fellow LAM sister volunteer leaders & advocates a few questions came up. Because this protein is not occurring in normal adult cell tissue but is found in many cancers is this a mutation that occurred in utero or do you have theories as to why it manifests this way in our communities? If it is an utero origination do you think testing could be developed one day to use this a potential genetic biomarker? Or if present be used as guide for an in utero treatment that could halt or delay or even prevent LAM or TSC in future generations?
A: This is a complicated question and we are continuing to investigate this along with understanding the true origin of the LAM cell.
One of our ideas is certainly that HMGA2 could be used as a biomarker. However, we have not initiated those experiments yet.
Q: What do you think the future research implications are for this since it does seem to go against some basic science hypotheses about how our diseases behave? Do you think there are screenings that could be done for existing agents or drugs that could be used in combination with rapamune or separately to target this protein? Or will it take an entirely new agent? Do you think the drug treatments that might be available to possibly attack this protein have more intense side effects and drawbacks than our current treatment or is it too soon to tell? I know because of side effects & immunosuppression that not everyone can tolerate current treatment regimens & drugs currently being tested.
A: We are at the stage where we are seriously thinking of the next step of translating these studies into drugs that could inhibit the HMGA2 pathway. As you point out, drug screening needs to be worked out to identify useful targets. This needs to be worked out and both known and unknown drugs will be used in such a screen. Since HMGA2 is expressed at such low levels in the adult, it is expected that side effects will not be widespread—although it is always dangerous to predict. Since our studies show that mTOR is not activated in 50% of LAM tumors, inhibitors of the mTOR pathway will have zero effect other than their general anti-proliferative effect in these tumors. Since our preliminary studies indicate that mTOR is only activated at the end of tumor formation, the usefulness of inhibitors of the mTOR pathway is limited and they will not be relevant for a cure of the disease.
Q: I know a few of you have been in this research game a long time, especially in both diseases. What was your initial reaction as a researcher when you happened to make these discoveries?
A: Although LAM was simply another example of a benign mesenchymal tumor expressing HMGA2 it was very exciting to see the presence of HMGA2 in the lung because this then provided insight into another potentially targetable pathway to treat the disease. Further excitement came from the control experiments, which clearly contradicted the standing hypothesis on the mechanism of LAM tumor formation. Therefore, our findings had identified a completely novel mechanism of LAM tumor formation and presented the possibility of a totally new set of potential drug targets.
Q: What inspired you to decide to spend time researching LAM & TSC? What motivated you to get involved in studying these diseases?
A: Jeanine sees patients with LAM and this has been a continued inspiration to explore the pathogenesis of the disease and identify new therapeutic options. In our center we see at least one new patient a month with the disease, which is motivation to continue working towards a cure. LAM and TSC are probably the best model systems to study benign, mesenchymal tumor formation. Any knowledge gained from these LAM studies would also be applicable to more prevalent tumors such as uterine fibroids, which one third of all women suffer from. Additionally, insight gained from tumor formation in LAM studies informs researchers as to the processes occurring in the more commonly known epithelial tumors (breast cancer) that also affects women.
Q: Another question was that mutations in TSC2/TSC1 are not present in sporadic LAM patients. Can you talk a little bit more about why that is and how your study findings further understanding in this area?
A: [Due to the complexity of this question, Dr. D’Armiento & Dr. Chada sent me the following response which I think ties together this question & everything else quite nicely.]
1. As you know, DNA (or gene, and in our case TSC2) encodes for RNA, which in turn is translated into protein (tuberin), the latter being the functional molecule within the mTOR pathway. With the exception of a couple of studies (including our present study), all the TSC2 studies have been performed at the DNA level and INFERRED the outcome on the tuberin protein. In contrast, our study analyzed the protein encoded by the TSC2 gene, tuberin, directly in LAM tissue.
2. One of the functions of tuberin is to modify the mTOR pathway. This does NOT mean that it is the only function of tuberin.
3. We discovered that in 100% of LAM patients, tuberin was present. We also showed that approximately 50% of the LAM patients had an activated mTOR pathway.
4. The story with kidney tumors, angiomyolipomas in humans & renal cell carcinomas in mice differs somewhat & we will not deal with this here for the moment.
INTERPRETATION of RESULT
1. A direct prediction of the two hit hypothesis (which is derived from experiments performed at the DNA level) is that there should be an absence of tuberin in LAM samples. However, tuberin was present in 100% of LAM patients & so the two hit hypothesis does NOT apply to LAM.
2. In 50% of LAM samples, the mTOR pathway is NOT activated. Therefore, activation of the mTOR pathway is NOT required or necessary for LAM pathogenesis.
3. Rapamycin, in patients without an active mTOR pathway, likely works as a general anti-proliferative agent.
RAMIFICATIONS of RESULTS
We have concluded the following on the basis of our direct studies on tuberin in LAM. Please remember; our conclusions apply to the vast majority of LAM but there may be some exceptions.
In LAM, activation of the mTOR pathway is irrelevant to LAM pathogenesis in 50% of patients. Other pathways, and we believe that we have identified one such pathway, play a much greater role in LAM pathogenesis. Therefore, it is no surprise to us that there are sporadic LAM patients with no mutations in TSC1/2.
The final question is why is there an increased incidence of LAM in familial TSC as compared to the normal population? We believe that having one mutated allele of TSC2 increases the probability of LAM by as yet an unknown mechanism. TSC2 haploinsufficiency impacts other genetic pathways that could lead to the observed increased incidence. This also implies other functionalities of TSC2.
Finally, we believe that when activation of the mTOR pathway does occur in the 50% of LAM samples, it is a latter event in LAM pathogenesis and that this activation does NOT occur through the two hit method since tuberin is present in 100% of LAM samples. The mTOR pathway can be activated in many other multiple fashions and each of these might well occur in different patients. As enticing as it was for the two hit hypothesis to be linked with the mTOR pathway because of the possible role of TSC2 in LAM, it would seem that these assumptions were not valid.
As you can see from the above, the science is complex, but hopefully this post & Dr. Chada & Dr. D’Armiento’s willingness to discuss their findings with me helps our community breakdown this science a little better & digest this finding a little easier.
As we’ve said before, this does NOT negate prior research, just gives us more to consider in unraveling the mysteries of both diseases.