Podcasts about rtks

BUFFALO, NY- May 22, 2024 – A new research paper was published in Oncotarget's Volume 15 on May 17, 2024, entitled, “GZ17-6.02 kills PDX isolates of uveal melanoma.” In this new study, researchers Laurence Booth, Jane L. Roberts, Ivan Spasojevic, Kaitlyn C. Baker, Andrew Poklepovic, Cameron West, John M. Kirkwood, and Paul Dent from Virginia Commonwealth University, Duke University School of Medicine, Genzada Pharmaceuticals, Texas Tech University Health Sciences Center, and University of Pittsburgh Cancer Institute defined the biology of GZ17-6.02 in UM cells and in parallel determined its interaction with irreversible ERBB inhibitors (afatinib, neratinib) and with the cytotoxic agent doxorubicin. “GZ17-6.02 is a novel compound, containing the synthetically manufactured components: curcumin, harmine and isovanillin and has undergone phase I safety evaluation in cancer patients (NCT03775525).” GZ17-6.02 has undergone phase I evaluation in patients with solid tumors (NCT03775525). The RP2D is 375 mg PO BID, with an uveal melanoma patient exhibiting a 15% reduction in tumor mass for 5 months at this dose. Studies in this manuscript have defined the biology of GZ17-6.02 in PDX isolates of uveal melanoma cells. GZ17-6.02 killed uveal melanoma cells through multiple convergent signals including enhanced ATM-AMPK-mTORC1 activity, inactivation of YAP/TAZ and inactivation of eIF2α. GZ17-6.02 significantly enhanced the expression of BAP1, predictive to reduce metastasis, and reduced the levels of ERBB family RTKs, predicted to reduce growth. GZ17-6.02 interacted with doxorubicin or ERBB family inhibitors to significantly enhance tumor cell killing which was associated with greater levels of autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5 or eIF2α were more protective than knock down of ATM, AMPKα, CD95 or FADD, however, over-expression of FLIP-s provided greater protection compared to knock down of CD95 or FADD. Expression of activated forms of mTOR and STAT3 significantly reduced tumor cell killing. GZ17-6.02 reduced the expression of PD-L1 in uveal melanoma cells to a similar extent as observed in cutaneous melanoma cells whereas it was less effective at enhancing the levels of MHCA. The components of GZ17-6.02 were detected in tumors using a syngeneic tumor model. “Our data support future testing GZ17-6.02 in uveal melanoma as a single agent, in combination with ERBB family inhibitors, in combination with cytotoxic drugs, or with an anti-PD1 immunotherapy.” DOI - https://doi.org/10.18632/oncotarget.28586 Correspondence to - Paul Dent - paul.dent@vcuhealth.org Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28586 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, autophagy, ER stress, GZ17-6.02, doxorubicin, afatinib, neratinib About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. Oncotarget is indexed and archived by PubMed/Medline, PubMed Central, Scopus, EMBASE, META (Chan Zuckerberg Initiative) (2018-2022), and Dimensions (Digital Science). To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Spotify - https://open.spotify.com/show/0gRwT6BqYWJzxzmjPJwtVh MEDIA@IMPACTJOURNALS.COM

BUFFALO, NY- November 8, 2023 – A new editorial #paper was #published in Oncotarget's Volume 14 on March 31, 2023, entitled, “Impact of SHP2 tyrosine phosphorylation on the development of acquired resistance to allosteric SHP2 inhibitors.” The SH2 domain-containing tyrosine phosphatase 2 (SHP2) is a ubiquitously expressed non-receptor protein tyrosine phosphatase, encoded by the PTPN11 gene. It is positively regulated by upstream receptor tyrosine kinases (RTKs) to activate downstream the RAS-ERK pathway. In this new editorial, researchers Giulia Franciosa and Jesper V. Olsen from the University of Copenhagen discuss potential leukemia therapies that effectively prevent adaptive resistance. Acute myeloid leukemia (AML) is a bone marrow malignancy characterized by a blockage of differentiation and an uncontrolled proliferation of myeloid hematopoietic progenitor cells. The internal tandem duplication (ITD) in the juxtamembrane domain of the RTK FLT3 is an oncogenic driver mutation that leads to constitutive activation of its tyrosine kinase activity. Consequently, FLT3 inhibitors that block its tyrosine kinase activity represent the targeted treatment option for patients with FLT3-ITD AML, often administrated in combination with induction chemotherapy. “Nevertheless, the short duration of remission urges the development of novel combinatorial therapies for FLT3-ITD AML.” Since 2016, several potent and selective allosteric, noncovalent SHP2 inhibitors have been developed and tested in clinical trials for solid tumors. A recent study reported the effectiveness of short-term treatment with the allosteric SHP2 inhibitor SHP099 as a single agent in clinically relevant mouse models of Flt3-ITD AML. This observation was in contrast with published data showing that allosteric SHP2 inhibition is only effective as combination treatment with inhibitors of other nodes of the RAS-ERK pathway. In a research article published by Pfeiffer et al., the Olsen's lab at University of Copenhagen showed that two commercial FLT3-ITDpositive AML cell lines (MV-4-11 and MOLM-13) developed adaptive resistance after prolonged treatment in vitro with the allosteric SHP2 inhibitor SHP099. “All in all, the findings by Pfeiffer et al. suggest that combined inhibition of SHP2 and mutated RTKs are effective in preventing adaptive resistance, but also highlight the need for development of more potent and effective SHP2 inhibitors and combination therapies for clinical applications.” DOI - https://doi.org/10.18632/oncotarget.28392 Correspondence to - Giulia Franciosa - giulia.franciosa@cpr.ku.dk Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28392 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, SHP2, PTPN11, tyrosine, phosphorylation About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

How to obtain accurate mapping data? RTK use without GCPs? Today's episode is brought to you by Drone U In-person Events. If you're looking to enhance your flight skills (and who among us doesn't need to enhance our flight skills), we are hosting our last in-person mapping training in November in beautiful Colorado. This is our three-day boot camp plus one-day Flight Mastery Training. We will be introducing and flying a variety of data acquisition types followed by discussion and instruction on how to process the acquired data. From Pix4d Mapper to Pix4d React, to Drone Deploy and Optelos. Students will go through a total of 7 exercises to master the workflow for various deliverables. The goal is to map and build models of this training's location. Join us today !! On today's show we discuss about RTKs, GCPs and mapping accuracy and conducting successful mapping missions. Join us for today's episode where we discuss mapping in detail and go over the need for using RTK, GCPs. Our question from John today is about knowing more on RTKs, GCP and mapping data accuracy. Thanks for the question John, there has always been the need for pilots to be mindful of the many parameters for obtaining great mapping data and in today's podcast we go over all of them. We address John's question on using RTK without GCPs and provide the process for executing a perfect mapping mission and gather accurate data. Tune in today to learn more ! Get Your Biggest and Most Common Drone Certificate Questions Answered by Downloading this FREE Part 107 PDF Make sure to get yourself the all-new Drone U landing pad! Get your questions answered: https://thedroneu.com/. If you enjoy the show, the #1 thing you can do to help us out is to subscribe to it on iTunes. Can we ask you to do that for us real quick? While you're there, leave us a 5-star review, if you're inclined to do so. Thanks! https://itunes.apple.com/us/podcast/ask-drone-u/id967352832. Become a Drone U Member. Access to over 30 courses, great resources, and our incredible community. Follow Us Site – https://thedroneu.com/ Facebook – https://www.facebook.com/droneu Instagram – https://instagram.com/thedroneu/ Twitter – https://twitter.com/thedroneu YouTube – https://www.youtube.com/c/droneu Timestamps: [01:55] Overview of today's episode on GCP, Mapping, data acquisition accuracy [03:33] Today's question on GCP, RTK and accurate data collection [06:20] Marking GCP and the process involved [14:01] Using an RTK unit and steps to obtain data accurately

Hi Everyone! In Part II of the Receptor segment, we'll discuss RTKs, NR, and some way that things can go wrong. Hope you enjoy and learn something, and please feel free to reach out to me: medtogether26@gmail.com with comments or feedback!

JCO PO author Alexander E. Drilon, MD, shares insights into his article, “Efficacy and Safety of Larotrectinib in Patients With Tropomyosin Receptor Kinase Fusion–Positive Lung Cancers” and the article's findings of the activity of larotrectinib in patients with advanced lung cancer harboring NTRK gene fusions. Host Dr. Rafeh Naqash and Dr. Drilon discuss drug development, testing for fusions, resistance mechanisms, and cancer metastases. Click here to read the article!   TRANSCRIPT Dr. Rafeh Naqash: Hello and welcome to JCO Precision Oncology Conversations, where we bring you engaging conversations with authors of clinically relevant and highly significant JCO PO articles. I am your host, Dr. Rafeh Naqash, Social Media Editor for JCO Precision Oncology, and Assistant Professor at the OU Stephenson Cancer Center.  Today we are excited to be joined by Dr. Alexander Drilon, Chief of the Early Drug Development Service and Medical Oncologist on the Thoracic Oncology Service at the Memorial Sloan Kettering Cancer Center and lead author of the JCO Precision Oncology article “Efficacy and Safety of Larotrectinib in Patients With Tropomyosin Receptor Kinase Fusion–Positive Lung Cancers.” Our guests' disclosures will be linked in the transcript.  Dr. Drilon, welcome to the podcast and thank you for joining us today. We're really excited to be discussing this topic with you.  Dr. Alexander Drilon: It's my pleasure and thank you for the invitation. Dr. Rafeh Naqash: For the sake of this podcast, we will refer to each other using our first names. So, Alex, you've led the development for some of these agents targeting NTRK. So it's really timely that you're coming onto this podcast to not just discuss this very interesting paper that you published in JCO Precision Oncology, but also the general landscape of NTRK. So could you briefly tell us about the history of the drug development process behind NTRK fusions, when it started, how you got involved, and where it stands currently? Dr. Alexander Drilon: Sure. So, as you mentioned, my background is in lung cancer, where when I came on as a fellow, there was a lot of excitement around EGFR and ALK, but then subsequently other oncogene drivers were also discovered and many of them were fusion. So, as we know, ALK in the fuse state is a driver of many tumors, as is ROS1 and RET. And interestingly, NTRK fusions share many similarities with ALK, RET, and ROS1 in that you have an intact kinase domain that's in the three prime position, it's fused to a different gene in the five prime position and basically describes oncogenesis.  And the beautiful thing about NTRK fusions is that they are widely found across many different cancers. And I like to think of these cancers in two major buckets. So there is a bucket for cancers that are rare where we see these NTRK fusions with a very high frequency. And examples here are your secretory carcinomas of the salivary gland and the breast, for example, more congenital fibrosarcoma, where the frequency exceeds 90% in some series, and then there are much more common tumors where the frequency is much lower. So lung cancer is an example where you find it in less than 1% of cases. There are some other tumors like GI cancers also where the frequency is low. And beyond these two major groups, we also see these NTRK fusion-positive cancers occur not just in the adult population, but the pediatric population. All of that thrown together means that it was a really great setup for exploring the activity and safety of targeted therapy in what we call a ‘basket trial' paradigm, where you design a trial and instead of selecting patients based on cancer type, you ignore cancer type and, of course, you accrue by an enrolling alteration, which in this case is the NTRK fusion.  Dr. Rafeh Naqash: Excellent. Thank you for that summary. It's interesting that just yesterday in my phase I clinic, I had an individual who was supposed to go on a certain study, and liquid biopsy came back and showed an NTRK fusion for a very odd presentation of a prostate cancer, which, again, got me thinking about the paper that you published trying to read about NTRK and then this happened and I got thinking about a bunch of other questions. But, for starters, though, from a receptor standpoint and I know you published on this in different journals, could you briefly tell, for the sake of the audience, describe the pathway and the tyrosine kinase signaling and associated resistance pathways that are concurrently acting in a different direction, perhaps, and also discuss briefly from neural development? I know the pathway, the NTRK gene or TRK gene as such is involved in different neuronal signaling aspects. Could you briefly touch on that? Dr. Alexander Drilon: Sure. And thankfully there are a lot of parallels with other things that perhaps some of the listeners are more familiar with. We'll start with the fact that it is a receptor tyrosine kinase, NTRK. It's a gene that encodes a receptor tyrosine kinase just like other receptor tyrosine kinases that may be fused such as ALK, RET, and ROS1. But remember also that other RTKs are EGFR, FGFR, which are also well known. The important thing to remember for NTRK is that you have three different genes, NTRK 1, 2, and 3 that encode three different proteins which are called TRK A, B, and C. And as you intimated, in the non-oncogenic state, these are very important for the development and the maintenance of the nervous system. And in the fused state, of course, similar to other fusions that we spoke about, the chimeric oncoprotein will drive downstream signaling and tumor growth and metastases. And in general, these cancers can be very reliant on downstream signaling in the MAP Kinase pathway but may also on occasion activate other downstream pathways like the PI3 Kinase pathway. Dr. Rafeh Naqash: And I know some of that could potentially play into resistance mechanisms for some of these first or second-generation NTRK inhibitors. From a fusion partner standpoint, the data that I came across that you're very well aware of is different fusion partners, and maybe some have a slightly better prognosis than some other fusions. But, in your practice and in your experience, does it matter what the other fusion partner is if the kinase domain is intact, meaning the signaling for the NTRK gene is intact? Have you seen any differences there from the other fusion partner standpoint? Dr. Alexander Drilon: From a patient-matching perspective, as long as you think the fusion is real, and by that I mean that you look at the report and you're sure the kinase domain is there and you're sure it's in frame, meaning connected well to the five prime partner so that the DNA strand is read through, the five prime partner does not play a major role in my deciding to give a TRK inhibitor or not. I would give anyone with a functional NTRK 1, 2, or 3 fusion a TRK inhibitor. Now, the data on whether or not select fusions do better than others is, I would say, still a little immature and perhaps conditioned by a few things. There are some of the cancers in the first bucket that we talked about, like the secretory carcinomas that harbor a recurrent event such as ETV6 NTRK3. And those cancers, in my experience in clinic, patients with those tumors can be on a TRK inhibitor for a very long time. And it's unclear if that's because of the exact fusion event or if it's because of the cancer type that might be more, say, genomically naïve compared to a gastrointestinal tumor, like a colorectal cancer with an NTRK fusion. So I hesitate to say that there are very strong and convincing data that if you have a particular five prime partner, you'll absolutely do better or worse. So, in the interim, I think the most important piece is just making sure that the event is real and actionable, and if it is, then you can give a TRK inhibitor. Dr. Rafeh Naqash: Thank you so much. I totally agree. And I think, for the sake of our listeners, as we see more and more sequencing being done on patients with cancer in the advanced stage setting especially, it's important to keep in mind when you have something that you can act on that has an actionable target that is FDA approved, then it's important to give the patient that option, especially in rare fusion events such as NTRK or TRK.   Now, you've touched upon this in your paper, but before we go into the details of the paper, specifically, I wanted you to briefly talk about the testing mechanisms which are important for some of these fusions and play into, for example, ROS1 ALK fusions also. Could you tell us what are the most appropriate ways to test for these fusions in patients harboring cancers, both from a tissue standpoint and from a blood-based assay standpoint? Dr. Alexander Drilon: This is a great question because if you don't have a test that's optimally poised to pick up an NTRK fusion, then you can't act on it. And a patient who would have benefited very durably from a TRK inhibitor won't get access to it. So there are different ways of testing for NTRK fusions, and I like to think of the central dogma here where you have DNA becomes RNA becomes protein because that really helps anchor the different types of assays that you might use. We commonly use next-generation sequencing of DNA, but even if you have a very good next-generation sequencing assay, that does have its limitations because there are some fusions that are structurally just difficult to pick up even with a great DNA-based NGS assay.  And for that reason, we and others have found that in tumors that have an equivocal NTRK fusion, or perhaps where you didn't find something but you really suspect that you missed something, particularly in cases where, historically, like congenital fibrosarcoma where you know there's a very good likelihood of finding NTRK fusion, we then reach for an RNA-based assay because at the RNA level, you've removed things like the intra-DNA based capture challenging. And so I think that from a nucleic acid standpoint, leveraging a test that looks both at DNA and RNA, maximizes the likelihood of finding this fusion. And just remember that there are different NGS assays in terms of the approach to design and some might be more Amplicon-based and that's less optimal, but the hybrid-capture-based ones tend to be better. The DNA and RNA tests can be done on tumors, and in blood, you could do a liquid biopsy. It's very hard to fish out RNA in blood given the current technology so we're still limited to circulating tumor DNA which shares the liabilities of doing DNA testing on a tumor sample. But if you find it and it looks real, then it's certainly actionable even if you detect an NTRK fusion with a liquid biopsy.  Now going back to the central dogma there, the third piece which we haven't touched on is protein. And there have been many papers published now on the utility of immunohistochemistry, and this helps you confirm that the TRK A, B, and C proteins are actually expressed. And what tends to happen is in many fusions, the chimeric oncoproteins strongly express as TRK A, B, and C that helps provide a complementary test or assay that confirms that you're dealing with something that is actionable.  So that is a very contemporary approach and a very thorough approach to looking for these NTRK fusions where you do DNA and RNA if possible. And if you still have questions, ask your pathologist to see if they can do Pan-TRK IHC. But depending on the resource environment that you're in, there are older tests like FISH which we use for ALK that can also find these fusions. RT-PCR which only finds particular events, these can detect NTRK fusions but really don't have the breadth and comprehensiveness as the other assays that we discussed like NGS.  Dr. Rafeh Naqash: Thank you so much, Alex, for that amazing summary of all the methods that potentially could help detect this rare but important event. From a therapeutic standpoint, now, taking a deeper dive into your very interesting JCO Precision Oncology paper that looked at larotrectinib data from a pooled analysis of two trials, a phase II and a phase I. Could you tell us a little background about these two trials, the patient population and what kind of data were you trying to evaluate? And then we can discuss some of the interesting results that you showcase in the paper. Dr. Alexander Drilon: It really helps as a background to realize that the initial approach to this was really on a basket trial where the programs for larotrectinib, which is a selective TRK A, B, and C inhibitor, and the other drug entrectinib, which inhibits ROS1 in addition to TRK, really accrued pediatric and adult cancers with NTRK fusions. And this paper pulls out the lung cancer subset and we'll discuss that in detail. But before getting into that, it's important to know that in the tumor agnostic data set of all patients with an NTRK fusion of any type, larotrectinib achieved a response rate of approximately 80%, entrectinib of approaching 60%, and disease control was durable with a median PFS for larotrectinib of approximately 28 months, and with entrectinib numerically, the number was lower at 11 months.   So with that background, this paper in JCO PO, in the interest of featuring the activity for lung cancers with NTRK fusions, pulled out 20 patients with NTRK fusion-positive lung cancers. And the punchline is that the activity was pretty comparable to that seen with a bigger data set. So the objective response rate was 73% and many patients had a partial response, 67% of the cases, 7% had a complete response, and really only a minority had primary progressive disease, 1 patient out of the 15 evaluable patients. These responses and clinical benefit overall were durable and the median duration of response was almost 34 months, with a median progression-free survival of almost 35 and a half months and an overall survival median of 40.7 months.  And just to talk about how that stacks up compared to other targeted therapies, this certainly is in the ballpark of some of the best ALK inhibitors that we have for ALK fusion-positive lung cancer. It's also comparable to osimertinib for EGFR mutant lung cancer. So we can confidently view TRK inhibition in lung cancers with NTRK fusions as a highly-active therapy.  Dr. Rafeh Naqash: Absolutely. I think you touched upon this earlier where in your cohort at least 50% of patients had central nervous system involvement, and it looks like larotrectinib does have CNS activity and benefit. Could you speak to the differences between potential entrectinib and larotrectinib from a CNS efficacy standpoint? And the second part of that question was going to be when you identify this fusion in patients, for example, with lung cancer, now, since TRK does have a role in neuronal development, do you think there is a role for closer CNS monitoring in these patients if they have not had brain metastasis identified because of the fact that they have an NTRK fusion? Is there some predilection for CNS involvement from a metastasis standpoint? It's just something that I've been thinking of over the last couple of days after I saw my patient who does have CNS involvement but with prostate cancer, which I have not seen in the phase I setting in all the prostate patients that I've come across. So what are your thoughts on that?  Dr. Alexander Drilon: These are great questions. In lung cancers with NTRK fusions, there is a proclivity for metastasis to the CNS. And thankfully, both of these TKIs, larotrectinib and entrectinib, do have coverage of the CNS. Now, from a design perspective, the initial thought was perhaps entrechtinib was more CNS-penetrant. But if you look at the overall response rates in patients with brain metastases and the intracranial response rates where you have patients with target lesions in the brain that you're able to measure; if you look across the aisle, entrectinib and larotrectinib have comparable results, with the objective response rate being in the order of 50% to 60% and the intracranial response rate being also in the order of about 50% to 60%. So at the end of the day, it appears as if, despite the previous hypothesis that maybe one drug would work better in the CNS than the other, we're seeing equally good effects with both drugs.  For the second question you asked, it's also a very interesting question because, like you mentioned, the TRK receptors play a role in nervous system development. But we have not observed a much higher frequency of CNS metastases in NTRK fusion-positive lung cancers or cancers in general that I know of, compared to cancers that are wild type for an oncogene or have other oncogenes. So what's more important really to think about when you sort of chew on the fact that these TRK inhibitors are involved in nervous system development are the potential side effects that you may see in patients that you treat with these TRK inhibitors. Dr. Rafeh Naqash: Absolutely. Now, from the therapy standpoint that you discuss here, duration of responses, objective responses that you saw in your analysis were very impressive for these patients with lung cancer. In your clinical practice if you see a lung cancer patient with this fusion and you treat them with larotrectinib or entrectinib, and they have, let's say, de novo CNS metastases that are asymptomatic, do you generally try the targeted therapy first and hold off, perhaps, brain directed therapy in that setting? Similar to what one would do with osimertinib perhaps or alectinib?  Dr. Alexander Drilon: Absolutely. It's the same paradigm because we know that we are seeing in a larger population of patients, just generally good activity, both extracranially and intracranially. The goal is to try to spare patients the extra side effects of doing radiation by only giving the TKI. And in practice, even outside of the trial, in patients that I've treated with CNS metastases, the activity has been very good. Dr. Rafeh Naqash: Thank you so much. Now, all TKI therapies have, unfortunately, resistance mechanisms that come up eventually, in my experience at least. What is your experience as far as understanding resistance mechanisms on TRK-based therapies and potential second options after that, whether it's second-generation TRK inhibitors or subsequent targeted therapies in this space? Dr. Alexander Drilon: Thankfully, this has been looked at extensively and I like to categorize resistance into two major groups. So there's a type of resistance which we call on-target resistance and another type which we call off-target resistance. In simple terms, cancers that acquire on-target resistance are still dependent on the NTRK or TRK pathway. And often what happens is, like with other oncogene-targeted therapy pairs, you see the acquisition of a resistance mutation in the kinase domain of NTRK 1, 2, 3 that either changes the dynamics of the kinase or sort of kicks the drug off of the binding site due to steric hindrance.  And for those patients, companies have designed next-generation TRK inhibitors that abrogate resistance, meaning they were designed so that they would work despite the presence of these resistance mutations. And a few of them include repotrectinib, talatrectinib, and selitrectinib that are thought to have activity, but there are many other newer ones that are currently being explored. I will say that there's proof of concept that has been published as well showing that patients who progress on a first-generation TRK inhibitor like larotrectinib or entrectinib who develop acquired resistance that's on-target can respond very well to a next-generation NTRK inhibitor. And while these aren't approved just yet, these are of course available in clinical trials. Now, the second major group is more problematic. This is off-target resistance. And when I describe this to patients, what I usually say is that the cancer sort of ‘phones a friend' and activates a second gene perhaps that isn't NTRK. And examples of that include KRAS or MET or BRAF, very well-known oncogenes in other contexts, but it leads to a reliance outside of the NTRK or TRK pathway per se, which still effectively reactivates the MAP kinase pathway. What to do in that situation? Well, there are select cases and there have been case reports published of patients who get a combination. Say if it's acquired MET amplification, you give a MET inhibitor with a TRK inhibitor and that combination can work. But in many other cases where you don't have access to a combination on a clinical trial or on compassionate use, then you really default to the standard of care for that cancer type. So if it's lung cancer and they've never had chemotherapy before, then it would be platinum-based chemotherapy, say with pemetrexed and a third drug, perhaps if they have lung adenocarcinoma.  Dr. Rafeh Naqash: Thank you so much. This is definitely an exciting field and exciting time to be in this space of drug development, and especially when we have so many interesting tumor-agnostic approvals that have come along in the last few years and more to come. And you've led a lot of this development with several other leaders in this field. So it was very nice discussing this with you, and hopefully, our listeners find it equally interesting and educationally relevant to what we see day in and day out as we perform more and more sequencing for patients with cancer and try to identify some of these rare or not so rare events that are targetable and can definitely change the course of a patient's therapy and outcomes. So thank you once again, Alex, for the discussion on this paper.  But before we end, we'd like to spend a couple of minutes trying to know about the investigator. So could you tell us a little bit about your career trajectory, how you started your fellowship perhaps, how you ended up in drug development, and how you've successfully contributed so much in this field to date? Dr. Alexander Drilon: Sure. So I'm originally from the Philippines, was born there, finished med school, and really wanted to come to the United States to sort of broaden my education and my residency program in internal medicine, then called St. Luke's Roosevelt under Columbia, had a program that sent people to rotate through Memorial Sloan Kettering Cancer where I currently work. So that was my first exposure with oncology. I fell in love with it and eventually became a fellow, fortunately, at Memorial Sloan Kettering. And I mentioned earlier that during that time I had subspecialized in lung cancer and there was a lot of excitement around targeted therapy for oncogene-driven lung cancer. And that was my point of entry. I saw these drugs work very well and I said that if I were in a position to develop newer agents, perhaps for other oncogenes where there wasn't anything developed just yet, that would be really cool. And that was my entry into the phase I world where things later on expanded really the tumor agnostic interrogation using the same principles that were familiar to me in the lung cancer world. And I think I've been very fortunate with the environment and the ability, especially with good in-house sequencing, to match many patients to these trials. And it's been wonderful to see several of these drugs approved. Larotrectinib was the sort of seminal tumor-agnostic approval of a targeted therapy for the first time by any regulatory body. And like you said, the hope is that we see several more of these. Dr. Rafeh Naqash: Awesome. That sounds like a very interesting, phenomenal journey that you've had, and a lot of it is also probably related to the kind of people that you met, mentors, and other people who helped you along the way. And then, of course, you've done a lot for other fellows and trainees in this space of drug development. So thank you again, Alex, for joining us, and thank you for choosing JCO Precision Oncology as a destination for your work. I look forward to interacting with you further subsequently and hopefully seeing more development in this space of novel therapies for fusions and other interesting targets in the lung cancer space.  So thank you for listening to JCO Precision Oncology Conversations. Don't forget to give us a rating or review and be sure to subscribe so you never miss an episode. You can find all ASCO shows at asco.org/podcasts.  The purpose of this podcast is to educate and to inform. This is not a substitute for professional medical care and is not intended for use in the diagnosis or treatment of individual conditions.   Guests on this podcast express their own opinions, experience, and conclusions. Guest statements on the podcast do not express the opinions of ASCO. The mention of any product, service, organization, activity, or therapy should not be construed as an ASCO endorsement.  Bio: Alexander E. Drilon, MD, is a medical oncologist specializing in the treatment of lung cancer. He is chief of early drug development service at Memorial Sloan Kettering Cancer Center. He has clinical expertise in lung cancer and early-phase clinical trials.   COIs Alexander Drilon Honoraria: Medscape, OncLive, PeerVoice, Physicians' Education Resource, Targeted Oncology, MORE Health, Research to Practice, Foundation Medicine, PeerView Consulting or Advisory Role: Ignyta, Loxo, TP Therapeutics, AstraZeneca, Pfizer, Blueprint Medicines, Genentech/Roche, Helsinn Therapeutics, BeiGene, Hengrui Therapeutics, Exelixis, Bayer, Tyra Biosciences, Verastem, Takeda/Millennium, BerGenBio, MORE Health, Lilly, AbbVie, 14ner Oncology/Elevation Oncology, Remedica, Archer, Monopteros Therapeutics, Novartis, EMD Serono/Merck, Melendi, Repare Therapeutics Research Funding: Foundation Medicine Patents, Royalties, Other Intellectual Property: Wolters Kluwer (Royalties for Pocket Oncology) Other Relationship: Merck, GlaxoSmithKline, Teva, Taiho Pharmaceutical, Pfizer, PharmaMar, Puma Biotechnology

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.03.527005v1?rss=1 Authors: Payne, L. B., Abdelazim, H., Hoque, M., Barnes, A., Mironovova, Z., Willi, C. E., Darden, J. A., Houk, C., Sedovy, M. W., Johnstone, S. R., Chappell, J. C. Abstract: The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRb) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases (RTKs) like PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRb (sPDGFRb) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRb variants, and specifically during tissue homeostasis. Here, we found sPDGFRb protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRb isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRb by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRb transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRb protein was detected throughout the brain parenchyma in distinct regions such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRb variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRb variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRb likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRb in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion -- critical processes underlying neuronal health and function, and in turn memory and cognition. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.514582v1?rss=1 Authors: Rybak, J. A., Sahoo, A. R., Kim, S., Pyron, R. J., Pitts, S. B., Guleryuz, S., Smith, A. W., Buck, M., Barrera, F. N. Abstract: The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anti-cancer therapeutics. Current therapeutics target the kinase domain or extracellular region of EGFR. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the EGFR TM dimer. We propose that the resulting disruption of native TM interactions disrupts the conformation of the kinase domain, inhibiting the ability of EGFR to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.01.322123v1?rss=1 Authors: Lin, C.-C., Wieteska, L., Poncet-Montange, G., Suen, K. M., Arold, S. T., ahmed, z., Ladbury, J. E. Abstract: Despite the kinetically-favorable, ATP-rich intracellular environment, the mechanism by which receptor tyrosine kinases (RTKs) repress activation prior to extracellular stimulation is poorly understood. RTKs are activated through a precise sequence of phosphorylation reactions starting with a tyrosine on the activation loop (A-loop) of the intracellular kinase domain (KD). This forms an essential mono-phosphorylated active intermediate state on the path to further phosphorylation of the receptor. We show that this state is subjected to stringent control imposed by the peripheral juxtamembrane (JM) and C-terminal tail (CT) regions. This entails interplay between the intermolecular interaction between JM with KD, which stabilizes the asymmetric active KD dimer, and the opposing intramolecular binding of CT to KD. A further control step is provided by the previously unobserved direct binding between JM and CT. Mutations in JM and CT sites that perturb regulation are found in numerous pathologies, revealing novel sites for potential pharmaceutical intervention. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.20.305532v1?rss=1 Authors: Chen, L., Shu, X., Chen, Q., Wei, T., Wang, X., Wu, Q., Wang, H., Zhang, X., Liu, X., Zhang, Y., Zheng, S., Huang, L., Xiao, J., Jiang, C., Wang, Z., Yang, B., Guo, X. Abstract: Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine (pTyr) sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation. Multiple receptor tyrosine kinases (RTKs) can trigger Rpt2-Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates 26S proteasome integrity and activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase non-receptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anti-cancer therapies. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.09.229716v1?rss=1 Authors: Kalogriopoulos, N., Lopez-Sanchez, I., Lin, C., Ngo, T., Midde, K., Roy, S., Aznar, N., Murray, F., Garcia-Marcos, M., Kufareva, I., Ghassemian, M., Ghosh, P. Abstract: The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs crosstalk has been a long-standing question, but answers remain elusive. Using linear-ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator, GIV, dissociates G-alpha(i)/beta/gamma trimers, scaffolds monomeric G-alpha(i) with RTKs, and facilitates the phosphorylation on two tyrosines located within the inter-domain cleft of G-alpha(i). Phosphorylation triggers the activation of G-alpha(i) and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cellular decision to go vs. grow. These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes. Copy rights belong to original authors. Visit the link for more info

Jane Antony, postdoctoral researcher, Stanford Medicine, Stem Cell Biology and Regenerative Medicine, delivers an important and thorough overview of the current state of cancer research and methods that may lead to promising new discoveries. Antony worked toward her Ph.D. through a unique joint program of the National University of Singapore and Imperial College London to bolster her study and understanding of cancer cell biology. She has been an active member of various research groups in Singapore and the UK that focus their efforts primarily on cancer biology, ovarian cancer, metastasis, signal transduction, and therapeutics. Her current work pertains to the identification of innovative therapeutic targets in invasive breast cancer illuminating differential signaling schemas in tumor heterogeneity. Antony is constantly curious, with a passion for understanding the link between metastasis and cancer stem cells. Antony discusses her current cancer research. She states since cancer is an intrinsic disease, whatever you use to target it will impact the normal healthy tissue as well. Generally, a disease's etiology, or cause, can be classified as intrinsic, extrinsic, or idiopathic. Antony's particular interest is in regard to metastasis and how cancer spreads. Antony states that many cancers can be removed surgically and can utilize therapies that shrink the cancer cells, but if it spreads to other organs it becomes more difficult to treat. Antony gives a detailed analysis of what is currently known about DNA as it relates to cancer growth. She describes the different types of cancers, how they metastasize, and typically where they travel to, or could. Additionally, she provides an in-depth overview of the current therapies such as radiation and chemotherapy. In regard to radiation therapy, it certainly has an effect on normal cells as well, and chemotherapy, unfortunately, becomes less effective over time and multiple treatments.  Antony talks about some of her work and areas of particular interest such as tumor suppressors. Understanding how tumor suppressors are involved in the development of cancer, specifically genes that regulate Receptor Tyrosine Kinases (RTKs) can quite possibly provide some important answers. A gene of great interest is the OPCML, which is present in healthy, normal cells but is switched off in cancer, which makes treatment very difficult with lowered chances for recovery. Understanding how this gene functions in cells is a critically important issue. Targeting genes that regulate RTKs can lead to possible gene-based cancer therapies for patients. Antony details other important developments in research, such as limiting dilution assays (LDA). LDA is designed to define an undetermined frequency of effector cells within a population. They are dose-response assays that enable detection of a positive or negative immunoresponse in each unique culture within replicates that vary in the number of actual responder cells tested. By calculating stem cell frequencies researchers can study how stem cells are evolving throughout treatment. Antony describes her findings in regard to stem cells and various types of cancers. She states that in blood cancers, the methods are somewhat different, in that bone marrow is removed and then reinstated after treatment has flushed out the affected area. Obviously, this is a treatment advantage of liquid cancers, for solid tumor cancers cannot benefit in the same way due to their form. And she talks about an assortment of other interesting trials that are underway that may provide further answers of great benefit to science and medicine. Antony is a postdoctoral member of the American Society for Cell Biology (ASCB), as well as a member of the EMT International Association, and an associate member of the American Association for Cancer Research. She holds a Doctor of Philosophy from the National University of Singapore, a Bachelor of Engineering from the National University of Singapore, and a Doctor of Philosophy from Imperial College of Science, Technology & Medicine.

On Thursday, 65 countries representing 83% of international aviation agreed to cap their greenhouse-gas emissions from international flights at 2020 levels from 2021 onward – in part by forcing airlines to offset emissions above that threshold, and MAYBE by funding programs that save forests and support sustainable agriculture around the world. A final decision on offset types, however, isn’t expected until 2018 Backgrond: The Paris Climate Agreement created a framework for keeping the global rise in temperatures below 2 degrees Celsius (3.6 degrees Fahrenheit) over pre-Industrial levels, but it left emissions from international flights in limbo – partly because their "international" nature made it hard to reach agreement on which countries to charge the emissions to. That changed on Thursday, when the International Civil Aviation Organization (ICAO), the UN agency charged with coordinating aviation regulation, including environmental impact, agreed to freeze net aviation emissions at 2020 levels beginning in 2021, and to force airlines to offset emissions above that threshold. The program, called “CORSIA” (Carbon Offsetting and Reduction Scheme for International Aviation), will be phased in, with a voluntary pilot phase running from 2021 through 2023, then a second voluntary phase from 2024 through 2026, and a final phase, running from 2027 through 2035 that is mandatory for all countries except the very poor. ICAO President Olumuyiwa Benard Aliu said that 65 countries had already signed on for the voluntary phase, and these countries together represent nearly 83 percent of total aviation miles, measured in "revenue tonne kilometers" (RTKs), which translate into one metric ton of load (human passengers or cargo) per kilometer traveled. Includes Interviews with Dutch environmental attorney Jos Cozijnsen and Arjun Patney, policy director of the American Carbon Registry.

Die akute myeloische Leukämie (AML) ist aus genetischer Sicht eine sehr heterogene Erkrankung. Rezeptortyrosinkinasen (RTKs) wie FLT3 sind in der Leukämogenese von zentraler Bedeutung. Durch Mutationen aktivierte RTKs sind allerdings alleine nicht in der Lage eine AML zu induzieren. Die Kooperation mit anderen Mutationen ist hierfür notwendig. Zu den am häufigsten gemeinsam auftretenden Mutationen in der AML gehören NPM1- und FLT3-ITD- (internal tandem duplication) Mutationen. Klinische Daten zeigen, dass eine FLT3-ITD die gute Prognose von NPM1-mutierten (NPM1c+) Patienten in Abhängigkeit des FLT3-ITD-mRNA-Levels in negativer Weise beeinflusst. Dies lässt auf ein pathogenes Zusammenwirken beider Genmutationen in der AML schließen, welches im Rahmen dieser Arbeit untersucht wurde. Dazu wurde basierend auf der humanen AML-Zelllinie OCI-AML3 mittels stabiler, lentiviraler Transduktion das erste zelluläre Modellsystem etabliert, das die relevanten Genotypen (NPM1c+/FLT3-ITD; NPM1c+/FLT3-WT) sowie unterschiedliche Verhältnisse von FLT3-ITD zu FLT3-WT (ITD/WT) im NPM1-mutierten Hintergrund modelliert. Zunächst wurde die NPM1-Mutation sowie die Funktionalität des FLT3-WT- und FLT3-ITD-Rezeptors in den nativen und transgenen Zellen bestätigt. Mit Hilfe des Zellmodells konnte gezeigt werden, dass Zellen, die beide Mutationen tragen, in vitro wie auch in vivo einen Wachstumsvorteil besitzen. Dieser vergrößerte sich zudem mit zunehmendem ITD/WT-Verhältnis. Ab einem bestimmten ITD/WT-Verhältnis konnte dieser Wachstumsvorteil in vitro mit einem FLT3-Inhibitor über eine gewisse Dauer gehemmt werden. Diese Ergebnisse könnten auf ein Zusammenwirken der beiden Mutationen bei der Leukämogenese hinweisen und eine Ursache für die schlechteren Überlebenskurven von Patienten mit beiden Mutationen und zunehmender FLT3-ITD-Last darstellen. Der insgesamt jedoch nur schwach ausgeprägte Phänotyp des etablierten Zellmodells erfordert zum eindeutigen Nachweis der funktionellen Interaktion von NPM1- und FLT3-ITD Mutationen ein alternatives Modellsystem. In diesem Zellmodell zeigten Zellen, die den FLT3-WT-Rezeptor überexprimierten, ebenfalls einen schwachen Wachstumsvorteil gegenüber nativen Zellen mit endogener FLT3-WT-Expression. Neben aktivierenden FLT3-Mutationen wie einer ITD, führen auch hohe FLT3-WT-Expressionslevel zur konstitutiven Aktivierung der FLT3-Kinase und verschlechtern die Prognose der Patienten. Deshalb wurde in dieser Arbeit mit der Untersuchung der transkriptionellen Regulation von FLT3, als mögliche Ursache hoher FLT3-WT-Expressionslevel, begonnen. In silico wurden im proximalen FLT3-Promotor Bindestellen für die hämatopoetischen Transkriptionsfaktoren (TF) PAX5 und MYB identifiziert. Mit Hilfe des Dual-Luciferase® Reporter Assay Systems wurden PAX5 als schwacher Repressor und MYB als Aktivator des Flt3-Promotors bestätigt. Auch der Transkriptionsfaktor CEBPA verhielt sich auf gleiche Weise als Aktivator der Flt3-Promotoraktivität. Eine Punktmutation im CEBPA-Gen, die aus zwei AML-Fällen bekannt ist, führte zu einer erhöhten Flt3-Promotoraktivität. Die Identifizierung weiterer mutierter, FLT3-regulierender TF und ihre Korrelation mit der FLT3-Expression sollen zukünftig tiefere Einblicke in die transkriptionelle Regulierung von FLT3 als Ursache der FLT3-Überexpression in AML-Patienten gewähren. Für eine Reihe von in AML-Patienten gefundenen Mutationen ist deren Rolle in der Pathogenese der AML noch unbekannt. Dazu gehören Mutationen in den Rezeptortyrosinkinasen DDR1 und DDR2. In der vorliegenden Arbeit wurden DDR1- und DDR2-Mutationen stabil in Ba/F3 Zellen und transient in HEK-293T Zellen exprimiert, um ihr transformierendes Potential zu untersuchen und diese funktionell zu charakterisieren. Transgene, DDR1- und DDR2-exprimierende Ba/F3 Zellen zeigten keinen transformierenden Phänotyp. Weitere Untersuchungen zeigten eine konstitutive Phosphorylierung der extrazellulären DDR2-Mutanten (G222R, M291I) in HEK-293T Zellen und eine Adhäsion von Ba/F3 Zellen mit wildtypischem sowie mutiertem DDR1-Rezeptor in Anwesenheit des DDR-Liganden Kollagen. DDR1- und DDR2-Rezeptoren sind bisher vor allem in soliden Tumoren untersucht. Weitere funktionelle Analysen sind notwendig, um ihren Stellenwert bei der Entstehung von AML zu erfassen. Diese Arbeit zeigt, dass Rezeptortyrosinkinasen in der Leukämogenese auf unterschiedliche Weise eine wesentliche Rolle spielen können. Da Rezeptortyrosinkinasen zudem wichtige Zielmoleküle für therapeutische Ansätze darstellen, sind sie von besonderer Bedeutung.

Die Grundlagen der Satellitenpositionsbestimmung sind den meisten wohl bekannt. Dennoch bieten die Funktionsweise und Betriebsbedingungen von GPS und Co. viele interessante und spannende Details. Z.B. wurde der künstliche Fehler von GPS abgeschaltet, damit das US-Militär erhöhte Positionsgenauigkeit im Golfkrieg erhielt. Dieser Vortrag führt in die Funktionsweise von GPS ein, und zeigt viele Erweiterungen zur Erhöhung der Genauigkeit, die von den Systemarchitekten nie vorgesehen waren. Dies führt bis zu modernen Verfahren für RTKs, die in Echtzeit Positionsbestimmungen im Millimeterbereich ermöglichen. Neben dieser Funktionsbeschreibung soll der Vortrag vor allem aber auch einen Überblick bieten, wie diese Hochgenauigkeit mit kleinen Tricks auch mit handelsüblicher Hardware und OpenSource-Software möglich ist und wieviel davon auch im Handy erreicht werden kann. Dass diese exakte Positionsbestimmung auch in Zeiten hochauflösender Satellitenbilder nicht nur dem Selbstzweck dient wird abschließend anhand möglicher Anwendungen aufgezeigt und vorgestellt.

Flt3 is the most recently discovered member of the platelet-derived growth factor receptor (PDGFR) subfamily of receptor tyrosine kinases (RTKs). It is expressed on early hematopoietic progenitor cells and is involved in their growth. Activating mutations of Flt3, such as internal tandem duplications (ITD) in the juxtamembrane (JM) region of Flt3, are among the most common genetic alterations found in patients with acute myeloid leukemia (AML). For this reason Flt3 is an attractive target for specific tyrosine kinase inhibitors, and a number of such compounds are currently being tested in clinical trials. However, the mechanisms of Flt3-mediated signal transduction are poorly understood. Src family tyrosine kinases (SFKs) are involved in the signaling processes of various RTKs, including those of the PDGFR subfamily . Most SFKs are expressed predominantly or exclusively in hematopoietic cells, and at least Hck and Lyn were found to be activated in AML patients. These observations indicate that SFKs could also be involved in Flt3 signaling. In this work the role of SFKs in signal transduction of the Flt3 receptor was investigated. A panel of Hck and Flt3 mutants was used in biochemical and biological assays to deduce the contribution of kinase activity, SH2 and SH3 binding domains and tyrosine phosphorylation status to Flt3-SFK interaction. The work presented shows that SFKs interfere with the maturation of wild type and Flt3 ITD receptors in human embryonic kidney 293 (HEK- 293) cells in a kinase-dependent manner. The SFK-mediated effect on receptor maturation is reflected by accumulation of the immature, intracellular form of Flt3 at the expense of the mature, plasma membrane-inserted form of the receptor. This effect of SFK kinase activity on receptor maturation is not limited to Flt3, because analogous results were also obtained for the Kit receptor, another member of the PDGFR subfamily. The demonstration of the role of SFKs in regulation of receptor maturation is novel and the exact mechanism underlying this effect requires further investigation. Using the HEK-293 cells it was also shown that Hck is able to phosphorylate Flt3 on tyrosine residues and to associate with autophosphorylated Flt3 in an SH2 domain-dependent manner. Hck-mediated tyrosine phosphorylation occurs in the JM region of Flt3. Tyrosine residues 589 and 591 in the JM region, when phosphorylated, were also identified as the docking sites for Hck. Although Hck phosphorylation and binding sites on Flt3 overlap, Hck-mediated phosphorylation of Flt3 is not sufficient for Flt3-Hck association. In contrast to the situation in HEK-293 cells, Hck neither interferes with Flt3 maturation nor phosphorylates Flt3 on tyrosine residues, and does not detectably associate with the Flt3 receptor in the hematopoietic murine cell line 32D clone 3 (32D cl.3). However, the level of the ectopic expression of Hck in 32D cl.3 cells was lower than in HEK-293 cells. This difference in Hck expression level probably accounts for both the lack of the Hck-mediated phosphorylation of Flt3 and the interference with its maturation in 32D cl.3 cells. The lower Hck expression level, however, does not account for the lack of detectable association of Hck with Flt3 in 32D cl.3 cells. Using biological assays in which activation of Flt3 can partially overcome the requirement for cytokine stimulation in 32D cl.3 survival and proliferation, it was shown that Hck is not involved in Flt3 signal transduction leading to Flt3-mediated cell survival and Flt3 ITD-dependent cell growth. Therefore, biological relevance of SFKs in Flt3 signaling remains unclear. In summary, although the binding and phosphorylation of Flt3 by Hck can be shown, the biological relevance of Hck in Flt3 signaling remains to be formally demonstrated. This research led to the novel finding that, at least when over-expressed or hyperactivated, SFKs interfere with the maturation process of RTKs. Various studies have shown that the ligandindependent activation of RTKs as well as their premature phosphorylation can interfere with maturation of RTKs. This study reveals that, by virtue of their phosphorylating of Flt3, Kit and possibly other RTKs, SFKs can regulate maturation of these RTKs and consequently alter their transport to the plasma membrane. Further studies are required to investigate the biological relevance of this function of SFKs.

The Eph receptors comprise the largest subfamily of receptor tyrosine kinases (RTKs) with important roles during neuronal development. Unlike other RTKs, these receptors can be activated by their membrane bound ligands, the ephrins. Recent evidence strongly suggests that Eph receptors and their ligands also contribute to synapse formation and synaptic plasticity in the postnatal brain. However, the details of the mechanisms still remain unclear. In order to better understand the role of EphB2 receptors during these processes in living cells, EphB2 receptors were visualized. Therefore, one variant of the enhanced blue, green, or yellow fluorescent protein (E(C/G/Y)FP) was fused to the receptor. For this, ExFP was inserted in one of three different positions of EphB2: the N-terminus (EphB2-N), a site close to the juxtamembrane region (EphB2-C1) and between two functional relevant domains of the cytoplasmic tail (EphB2-C2). The different EphB2-ExFP receptors, EphB2-N, EphB2-C1 and EphB2-C2, were exogenously expressed in cell lines and showed intense fluorescence at the plasma membrane, comparable to other described transmembrane ExFP fusion proteins. Biochemical methods were used to test functionality of the different EphB2-ExFP proteins concerning tyrosine phosphorylation and interaction with known proteins, such as NR1 and GRIP2, and showed no obvious impairment. Surprisingly, however, the cluster behaviour of the EphB2-ExFP variants transiently expressed in neurons was different, only the EphB2- C1 proteins revealed a proper cluster formation after ephrinB stimulation, indicating that the ExFP insertion at the N- and C-terminus impaired the clustering. In order to study the dynamics of trafficking, insertion and cluster behaviour of fluorescently tagged EphB2 receptors in living neurons, hippocampal cultures were transfected with these constructs. EphB2-C1 expressing neurons revealed two pools of fluorescent clusters, one pool was static, representing EphB2 receptors at the plasma membrane, whereas the other pool was trafficking along neurites, presumably reflecting EphB2 proteins in transport vesicles. In addition, the subcellular distribution of these receptors was analyzed, revealing that e.g. EphB2 clusters are present at the tips of filopodia and in growth cones. Filopodia are highly dynamic structures, which explore the environment and therefore have to extend and retract a lot. Our group recently described a new mechanism of how an adhesive Eph-ephrin interaction between filopodia of immature growth cones and EphB2-expressing cell lines can be turned into a retraction response by bi-directional EphB/ephrinB-triggered trans-endocytosis. Intrigued by these findings, we were interested whether bi-directional transcytosis also exists in mature hippocampal cultures, presumably being involved in the dynamics of filopodia. The present thesis could show that after contact of mature neurons with cells, most-likely glial cells, the exogenous expression of fluorescently tagged EphB2 receptors in neurons induces a retrograde transcytosis into the interacting neighbouring cell. This reverse transcytosis of EphB2-C1 proteins was often followed by persistent retraction of the neuronal protrusion. This could have a potential role in axon pruning or in morphological plasticity in mature neurons, thereby adjusting the proper connectivity. In a second approach, thy1-EphB2-C1-EYFP transgenic mice were generated to gain insights into the in vivo behaviour of these fluorescently tagged proteins and to have a closer look in the dynamics and cluster behaviour of EphB2 receptors during synaptogenesis and in spines of more mature neurons. Contrary to our expectations, both the fluorescent intensities and the distribution of the EphB2-C1-EYFP proteins in these transgenic mice were not bright nor sparse enough for conclusive imaging experiments.

In der vorliegenden Arbeit wurde die Funktion der Rezeptor Tyrosin Kinase (RTK) FGFR4 (Fibroblast Growth Factor Rezeptor 4) in der Tumorentwicklung untersucht. Der FGFR4 besteht aus einer extrazellulären ligandenbindenen Domäne, einer einspännigen Transmembrandomäne und einem intrazellulären Bereich, der neben zwei Kinasedomänen auch eine Reihe von Bindungsmotiven für Adapterproteine mit und ohne enzymatische Aktivität enthält. Die Tyrosinkinase Funktion des FGFR4 wird durch lösliche Liganden, die FGFs, stimuliert. Die häufig starke Aktivität des FGFR4 Gens in Tumorgeweben lässt eine Funktion des FGFR4 in der Tumorentwicklung vermuten. Im Rahmen der vorliegenden Arbeit wurde zum ersten Mal eine Mutation in der Transmembrandomäne des FGFR4 nachgewiesen, die zu einem Austausch der hydrophoben Aminosäure Glycin gegen die hydrophile, stark geladene Aminosäure Arginin an Position 388 führt (Gly388Arg). Diese Mutation ist homolog zu der seltenen GlycinIn der vorliegenden Arbeit wurde die Funktion der Rezeptor Tyrosin Kinase (RTK) FGFR4 (Fibroblast Growth Factor Rezeptor 4) in der Tumorentwicklung untersucht. Der FGFR4 besteht aus einer extrazellulären ligandenbindenen Domäne, einer einspännigen Transmembrandomäne und einem intrazellulären Bereich, der neben zwei Kinasedomänen auch eine Reihe von Bindungsmotiven für Adapterproteine mit und ohne enzymatische Aktivität enthält. Die Tyrosinkinase Funktion des FGFR4 wird durch lösliche Liganden, die FGFs, stimuliert. Die häufig starke Aktivität des FGFR4 Gens in Tumorgeweben lässt eine Funktion des FGFR4 in der Tumorentwicklung vermuten. Im Rahmen der vorliegenden Arbeit wurde zum ersten Mal eine Mutation in der Transmembrandomäne des FGFR4 nachgewiesen, die zu einem Austausch der hydrophoben Aminosäure Glycin gegen die hydrophile, stark geladene Aminosäure Arginin an Position 388 führt (Gly388Arg). Diese Mutation ist homolog zu der seltenen Glycin dem Wildtyprezeptor (Gly388) und der Rezeptormutante. Die in vitro Kinase Assays der MAP-Kinase ERK2, die nach FGFR4 Stimulierung in L6 Myoblasten aktiviert wird, bestätigen diese Ergebnisse. In der Fähigkeit zu migrieren, unterschieden sich Brustkrebszellen, die stabil den mutierten FGFR4 exprimierten, dagegen deutlich von vergleichbaren Zellen, die den Wildtyprezeptor exprimierten. Daneben ergab die cDNA Array Analyse in den oben genannten Brustkrebszelllininen unterschiedliche Expressionsstärken für eine Reihe von Genen, die in der Tumorentwicklung eine bedeutende Rolle spielen. Des weiteren gelang es mit einem Fusionsprotein zwischen der extrazellulären FGFR4 Domäne und dem Glutathion-S-Transferase (GST) Protein eine ungewöhnliche Funktion des FGFR4 in der Zelladhäsion nachzuweisen. Nur wenige RTKs waren bis dahin als Vermittler von Zelladhäsion bekannt. Im Rahmen dieser Arbeit wurde gezeigt, dass verschiedene Zelltypen exklusiv auf Zellkulturplatten, die mit dem FGFR4-GST Fusionsprotein beschichtet sind, adherieren können. Im Gegensatz zu früheren Befunden ist dieser Prozess abhängig von zweiwertigen Kationen und kann nicht durch einen Überschuss von löslichen Heparin blockiert werden. Weiterhin konnten die spezifische Bindung von zwei Proteinen, deren Identität noch unbekannt ist, an das FGFR4-GST Fusionsprotein nachgewiesen werden, so dass von einer direkten Wechselwirkung zwischen der extrazellulären Domäne des FGFR4 mit weiteren Molekülen ausgegangen werden kann. Die Untersuchungen ergaben außerdem Hinweise auf eine Intgrin-vermittelte Signalkette, die während der Adhesion an FGFR4-GST induziert wird. Es wurde sowohl die Zunahme der Tyrosinphosphorylierung der “Focal Adhesion Kinase” (FAK) als auch die Aktivierung der MAP-Kinasen ERK2 und JNK gezeigt. Die hier erarbeiteten Daten ermöglichen also, eine Funktion des FGFR4 als morpho-regulatorisches Protein zu diskutieren. Darüberhinaus erlauben die in dieser Arbeit vorgestellten Ergebnisse erstmals eine Diskussion über die Funktion von Sequenzvarianten im FGFR4 in der humanen Pathogenese und insbesondere in der Tumorprogression. Sie zeigen, dass das vererbte FGFR4 Arg388 Allel mit einer schlechten klinischen Prognose in Brust- und Darmkrebs assoziiert ist und weisen auf einen Mechanismus hin, in dem der klinische Verlauf von Tumorerkrankungen durch vererbliche Parameter moduliert wird.