This is ATLAS.ONE (a high speed high resolution biochemical imaging platform)

Project ATLAS

In 2018, we decided to invest capital funds provided by the MRC and the MRC-DBT with the aim to make our technologies more accessible to the biomedical researcher laying down also the possibility to deliver advanced biophysical assays at high throughput [REF1] with a focus on 3D cultures. Why ATLAS? I often code-name internal projects, possibly with evocative names that might capture the pathos of the project. As these are capital investments to strengthen specific areas that will be essential for our long-term applications, I named this investment ATLAS, as the Titan that was condemned to hold up the heavens on its shoulders. In our case, I am building the base for two microscopes that will support my research projects, and support those of the others working at the MRC CU, in the longer period.

This is ATLAS.ONE

Here, I will briefly introduce ATLAS.ONE, the first of the two microscopes we have started to develop. The aim is to develop high spatiotemporal and biochemical resolution with a imaging platform that could be readily accessible by a non-expert user. We are testing the solid-state FLIM (Fluorescence Lifetime Imaging Microscopy) camera PCO.FLIM by PCO for video-rate biochemical read-out of genetically encoded probes or protein-protein interactions.  This is the commercial incarnation of part of my PhD work [REF2REF3] so brilliantly developed and delivered by PCO (no commercial conflict). After considering different possibilities to gain some resolution to better discriminate cellular compartments, we decided to integrate this platform with a simple SIM (structured illumination microscopy) setup based on LCoS spatial light modulators. Biochemical perturbations will be implemented with a CellASICS microfluidic platform. This is a capital investment and we will first focus on methodological advancements, however, we will deploy this platform to characterize genetic and non-genetic heterogeneity in cancer cell lines. While we will look for external funding, we’ll start working on KRAS-dependent signalling pathways and metabolic pathways.

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Who is involved?

You are welcome to get in touch if you wished to coordinate developments or to use this platform – once established – either with own resources or common grant applications. Currently, ATLAS.ONE is supported by the MRC and the MRC-DBT for capital funds and the following people for development and applications.

Andrew Trinh and Alessandro Esposito (MRC Cancer Unit), developing the system and applications together with Christian Frezza and Annie Howitt (MRC Cancer Unit), developing single-cell metabolic assays.

Guy Hagen, University of Colorado, to collaboratively develop SIM

Gerhard Holst, PCO to advise on camera integration.

 

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[Open hardware] A safe laser by-pass

Well, I remember when I started this business, a beam stop was done with a recycled block of lead and reflections stopped with carton boxes 😉 Brown boxes, black carton catches fires, of course (tell this to my undergrad-self). Not any longer, of course!

About ten years ago, I started the procurement and development of my first two-photon microscope. For the first time, I was directly responsible of laser safety and I had to take decisions about how to build a system that was safe for a user facility in a biomedical research institute. As I was coupling commercially sourced systems (Leica SP5, Chameleon Vision 2 and Pulse Select) and I was not planning much customization for the excitation path of this instrument (I heavily develop assays and detection), I opted to fully enclose the laser in lens tubes. The resulting system is safe, stable, and no more difficult to align compared to other enclosures.

I think that enclosures around the complete table might make sense in many instances, particularly when compartmentalized in sub-sections, but this is the system that worked best for me at the time. One solution I wish to share, is a bypass for the Pulse Picker we had used to develop spectrally resolved FLIM utilizing smart SPAD arrays (detectors that integrate photon counting electronics with them).

20181112_184730As I start planning replacement of this systems, I wished to share this design, in case some of you might find it useful. In the image on the left, you can see the Ti:Sapphire on the top, the pulse-picker on the right and the first enclosure by Leica used to steer the beam to their in-coupling optics (bottom-right).

In the middle, the laser bypass we utilize to direct the laser through the pulse-picker or around it.

In the image below, you see a close-up photo of the by-pass. The black box with the rectangular aluminum cover is the Leica spectral flattener used to reduce power of the Chameleon Vision at the peak wavelength. One of the few customization I needed here was simply to have a hole on a Thorlabs SM2 lens tube to accommodate this filter. This is screwed in a C4W-CC cube that can host a movable turning mirror with high reproducibility. The alignment of the microscope without the pulse-picker is done with the pair of mirrors provided by Leica. The alignment of the Pulse Picker is done with the kinematic mirrors visible on the left (M1 and M2). I placed a light-block behind them just in case one would become lose or to block the small amount of light transmitted through them. A kinematic cube is used to host ultrafast beam sampler by Newport to direct a small fraction of light to the Thorlabs PIN diode I use to feed the electronics of the pulse picker. In front of the PIN diode I have an xy-translating cage element. An empty four-way cube is used to allow the laser beam to pass from top to bottom (bypassed) or from left to right (coupled pulse picker). The aluminum block tagged as L1 is just a cover for the C4W-CC when empty.

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At the output of the pulse-picker, you see the mirror image of this bypass (on the right) and the two steering mirrors by Leica (the cylindrical towers). On the far right of the picture there is the in-coupling optics by Leica, preceded by two diagnostics ports.

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Below, you can see a close-up picture of this part of the coupling. Because of the layout, I needed to add one extra mirror (top left) and aiming to isolate users (placed on the top of the image) from accidental damages of the in-coupling optics, I added a light barrier.

Both diagnostics ports are based on a 4-way kinematic cube from Thorlabs hosting Newport beam samplers. The first port is used to sample the pulses after the pulse-picker and to feed our FLIM electronics. The second has two scopes. First, for course alignment of the system. I have two irises in the system that are aligned when the laser is aligned (roughly) to the in-coupling optics of Leica.

I usually remove a cover at the exit of this diagnostic port and use a fluorescent card to verify alignment, but in this picture you see the fiber coupling a spectrograph we occasionally use to diagnose faults of the laser.

 

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The alignment is simpler that it seems. First we start with a microscope that is fully aligned without pulse-picker as per normal operations. Then, when we need the pulse picker, we insert the two turning mirrors (L1 and R1). We do this with the laser off and with the pulse-picker crystal retracted (coarse alignment) or protected by an alignment card (fine alignment). M1 and M2 are then used to align the beam with the crystal. Then we align the PIN diode and proceed with the fine alignment of the pulse-picker cavity.  Once this is done, we align the cavity with the microscope utilizing M4 and M5. For course alignment, the signals from the two diagnostics ports is very useful until some signal is picked on the microscope monitor, after which the final fine tuning of all the optics can proceed.

Be aware, alignment of Class 4 lasers can be dangerous. Therefore, do your own risk assessments and think carefully about the logistics of your system. Now that I am starting to consider the redevelopment of the system, I thought to share these notes with you, hoping that it could be of some use.

Sharing is caring: an open access FLIM trial

Are you interested in cell biochemistry, but in single living cells, organoids or tissues? Is there a Western blot or IP you wished to do on a living sample? Or did you wish to see where in a cell a protein-protein interaction occurs.

Well, if you are interested in quantifying a ligand concentration, a post-translational modification, a protein-protein interaction, chromatin states, oligomerization of proteins, you might be interested in FLIM or FRET, but you might not be in your comfort zone to set-up or execute such assays. 

The specialist expertise and instrumentation required to perform fluorescence lifetime imaging microscopy (FLIM) is often a barrier to adoption of quantitative biochemical imaging techniques. The same can be true, although at a lesser extent, for intensity-based measurements of FRET.

Well, we have the expertise and we have the instrumentation. Not just this, but today, instrumentation and data analysis is becoming simpler and simpler. During 2019, we are going to trial a system by which we can support you for setting-up and test FLIM/FRET experiments. We have limited resources and, therefore, we will open only a few experimental sessions to start with, but there will be no strings attached. No fees, no authorship to include in that paper you really care.

Although we still have to setup the “Sharing is caring” trial, feel free to inform us about your interest. Initially, projects will be selected at our discretion, with priority given (but not confined) to cancer-related work and work with a potential to impact public health in the short or long period.

SOP – Ti:Sapphire / Leica SP5 alignment

This SOP is published only for a social media discussion. The author does not take any responsibility for the utilization of this procedure. The system discussed here is a customized two-photon microscope, based on a Coherent Chameleon Vision 2 and Leica SP5. The optical path is fully enclosed and the SOP is written for maintenance. 

Basic rules

1.       Align laser with a lid room (smaller iris, smaller damage to the eye)

2.       Never align eyes with the height/direction of the laser beam

3.       Use the most appropriate personal protective equipment such as goggles and a white lab coat

4.       Perform laser alignments with the least number of people present in the room. Ideally, alignment is a 1 person job and a colleague is aware you are performing this task, within core hours

5.       Use devices such cards, cameras and viewers to visualize the laser beam

6.       Take short breaks every ~45 minutes of work. Do not continue alignment if too tired. Alignment of optics can be a stressful and lengthy procedure; try to identify the right moment to take a long break to relax

7.       Alignment is carried out only by authorized users

PPE for Ti:Sapphire laser

          VC5 IR card viewer from Thorlabs. WARNING: card viewers reflect part of the laser beam. Therefore, they must be used with caution, strictly using protective goggles, directing reflection away from the eyes

          Hand-held IR viewer from Newport. WARNING: hand-held IR viewers limit dexterity and must be used always with protective goggles.

          LG9 Amber lenses from Thorlabs. OD5+ on the 720-1090nm range; OD7+ on the 750-1064nm range. WARNING: goggles never fully protect from direct high power laser beam.

List of authorized users

Alessandro Esposito (MRC Cancer Cell Unit)

Coherent’s field engineer can align the laser under their own responsibility. Coherent’s field engineer can align the beam path until after the Pulse-Picker. The rest of the optical path must enclosed at any time or isolated with a beam stop.

Leica’s field engineer can align the complete beam path under their own responsibility with the exception of the Pulse-Picker. Alterations of the beam path have been discussed with Leica representatives.

Standard Operating Procedure

Room preparation

1.       Show warning at the door

2.       Lock the door

3.       Switch on the system as needed (shutters ON)

4.       Wear PPE as appropriate

5.       Open the beam path as needed (keep lens tube arriving to the scan-head until the last moment)

6.       If a large section of the beam path is opened, always block the laser beam with the beam stop after the optical element that is aligned in order to avoid the laser beam being reflected in dangerous directions (eye, skin, fire hazard) when misaligned

Beam alignment

7.       Always activate laser shutter when the beam is not undergoing alignment

8.       Apply #6 every time a section of the laser beam is aligned

9.       Start laser alignment, proceed with pairs of mirrors from the position closer to the laser up to the scan-head, trying to operate the laser beam within a central part of the mirrors

10.   Always ascertain that all optomechanics is stably connected to the optical table and that no optical device can fall, tilt, flip…

11.   Re-aligned section should be covered (at least temporarily) while progressing towards the scan-head

12.   When arrived at the EOM, remove the device (Leica’s shutter and half-plate may be removed as well). WARNING: the entrance window of the EOM is located within a brass cavity. Upwards reflections of the laser are possible.

13.   Using irises, make sure the laser beam is parallel to the table

14.   Reposition the EOM, coarsely aligned to the laser beam. WARNING: after the EOM there is a periscope. Use a beam-stop before the periscope, beam reflection towards undesired direction is otherwise possible.

15.   With a power meter, measure power of the laser before the EOM. Relocate the power meter after the EOM and iteratively maximize power through the power meter with the EOM in “high” state.

16.   Coarsely align the periscope if necessary, then reintroduce Leica’s shutter and half-plate if previously removed. BE SURE the periscope is locked to the optical table in a stable manner.

17.   Remove lens tube and MFP cover.

18.   Install Leica’s alignment tool on the scan-head

19.   Iteratively align the front iris of the alignment tool and the back aperture of the alignment tool.

20.   WARNING. During the iterative alignment of scan-head, PPE is usually hindering an already lengthy procedure. Avoid removing PPE. Check actions to be taken.

21.   When the two apertures are aligned, start scanning trying to see fluorescence from a bright sample on the screen. Keep adjusting alignment and MFP screws until alignment is completed.

Preparing the room to normal operation

22.   Close the optical path. Before securing all covers and panels, check that the alignment is still ok.

23.   Secure all safety panels

24.   With the laser ON, shutter OFF and during scanning, verify with the IR viewer that no beam is exciting the enclosed laser path.

25.   Remove safety warning on the door and operate equipment as normal.

NyxBits and NyxSense? What?!

NyxSense&NyxBits paper here.

800px-Arte_romana,_statuetta_di_nyx_o_selene,_I_secolo_acI am not fond of new achronyms or ‘cool’ names, but then… guilty! you got me, I am contributing to the proliferation of four letters acronyms and fancy names like others! Lately, I have introduced a new one, HDIM as for Hyper-Dimensional Imaging Microscopy. But that is another story, and in a Supporting Note of that pre-print we explain our choice.

Earlier, we created the pHlameleons with the friend, my group leader back then, Fred Wouters. Well, first it was the Cameleon, the famous calcium reporter by the great Miyawaki and Tsien, brilliantly referred to as Camaleon because it is a protein that ‘changes colour’ upon binding calcium (Ca). Then it was the Clomeleon by Kuner and Augustine, as it senses cloride ions (Cl) rather then calcium. With all due respect for the authors, I must admit I did not love that name at first. Indeed, as we were deriving a family of pH sensors from yet another creation of Miyawaky (the CY11.5), we started to joke that we should have called this family of sensors the pHlameleons. Months after months, a joke ended up in a title of a paper, to be adopted as the name of these pH sensitive proteins. So, let’s not take ourselves too seriously too often. Sometimes we pick names for a bit of branding, other times to make our assays less heavy with too many technical terms, and other times, let’s just have fun with words (Clomeleon now for me is a great name, but I routinely joke about pHlameleons!).

Now that you know the little funny story about the pHlameleons, it is the turn of NyxSense and NyxBits. NyxSense is a software dedicated to multiplexing of FRET sensors. NyxBits are the components to create a multiplexing platform, a number of fluorescent proteins of distinct Stokes shift that can report, through their fluorescence lifetime, biochemical reactions probed via FRET with the use of dark/darker acceptor chromoproteins. A huge effort for us that took several years to bear fruit. Why Nyx?

During the revision of the drafts, colleagues found the manuscript a bit too technical and difficult to read. Thus I went back to pen and paper,  google and wikipedia, to find a name that could help us to refer to this sensing platform with a single word rather then a sentence. Greek mythology always provides great inspiration and eventually, I discovered Nyx the primordial goddess of the night (Nox in the Roman mythology). With Erebus (personification of darkness), Nyx gives birth to Aether (personification of the upper air and brightness), Moros (deadly fate), Moirai (destiny) and Thanatos (death). Then, I felt that this short name, Nyx, is intimately connected with our work for three reasons.

First, Nyx seems to link darkness and light, the day and night, a nice analogy with our bright donor fluorophores and dark acceptors. Second, Nyx is related to death and fate. We created the NyxBits and NyxSense to study cell fate, and our first application is cell death responses to an anti-cancer drug. Third, Nyx is a goddess and as I am really committed to gender equality at work (not just by picking names of fluorophores), it felt a little bit in tune with what I do, to honour a female deity.

But do not take these reflections too seriously – I do not – after all I needed just a simple name for a very complex sensing platform. As there is no way for me to tell the reasoning behind the names in the manuscripts, I thought to share with you why we picked NyxSense and NyxBit, light-heartedly.

 Now starting project Atlas… we’ll speak about this another time! 🙂

Embrace your public speaking anxiety

About a decade ago, I went to a PI during a retreat to ask a question. Nervously, but politely, he asked me to be left alone as he was rather anxious for a talk he was about to deliver.  A few hours later, a PhD student at the time, I was freaking out for my own talk, but it was comforting, in a way, to see that an established scientist I highly regarded and I had considered rather self-confident was in a similar state-of-mind.

Comforting? Why not scary? Would you never get rid of public speaking anxiety? I am no anxiety coach and, for that, browse around. However, I wished to share my own experience as it might be useful for students. I now noticed I am that ‘senior’ scientist at that retreat (or something similar) and that junior colleagues might misunderstand my confident speaking in public as evidence of no-stress, no-shyness, a gift from birth. So, even though your solution might be a different one, here I tell you which was mine.

Be prepared! Be prepared? (take 1)

Trivial, isn’t it? I am not going to give practical suggestions here, except set yourself comfortable deadlines. With experience, you will be able to work on a talk until a few minutes before delivery, but earlier in carrier, you have to prepare all your material far in advance. However, even very experienced academics and businessmen when facing more unique scenarios work hard to prep a meeting and give this enough time and resources.

Be prepared! Be prepared? (take 2)

Perhaps, the most difficult thing you might find, it is to commit to a deadline, after which you have to be ready. But, here the challenging bit, even if you feel still unprepared (and some people may never be able to shred off that feeling) or if you are actually unprepared because you miscalculated something, you have anyway to commit to the next difficult bit, be mentally and physically prepared, something you might be completing neglecting. Deadlines are deadlines and the starting time of your talk is unmovable. Therefore, start to mature a process and to understand how long you need to be ready before a talk. Some people is a natural and need no or little preparation. Other people need time: never underestimate how long time you need. Most of my following comments are about this stage of preparation. The bottom line, when the deadline strikes, be sure you are ready and if you are not, do not allow doubts to undermine the next phase of preparation.

Commit physically: water and energy

During a stressful moment, your physiology will be heavily altered and you might lose control. So, think how not to. Personally, before a talk I try to drink lots of water to ensure I will be properly hydrated, and I also make sure I have water available during the talk. Once I didn’t, and I was not well. I coughed though all my talk and it was not a very ideal situation. Also, be sure you have energy, so a bar of chocolate or a juice, can help. Ah… ok, is this obvious?… pay attention – water in > water out. As basic as it seems, be sure you went to the toilet at the latest opportunity before the event. You do not want to be dehydrated, but even not to be distracted by your bladder while on stage.

Mind you that this is even more true when you have very long days, such as more articulated interviews or conference commitments.

Commit physically: oxygen

Breathing, for me, is the next most important issue. You might find yourself in need of oxygen after a few slides and attempting to do the world record in apnoea while speaking in public. You could pass through an entire 20 minutes presentation incapable to breath properly, increasing your level of anxiety at each slide. You are in front of an audience, it could be two people at an interview, or a thousand people in a theatre, if not a million in TV. However, giving a good breath permitting your lungs to be completely emptied and filled with fresh air takes a few seconds. This can be easily concealed in a transition between two slides, or during a question. And… if you cannot conceal it… do it anyway, 5 seconds spent silently breathing properly will be immediately forgotten by your audience, but a poorly delivered 20 minutes talk will be remembered.

Once again, get ready for it. First of all reflect on your breathing habits, far away from a talk. If you give enough thoughts about the issue, whenever you will struggle, a mental trigger will snap and make you aware of the occurring issue for you to take action. More importantly, if issues in breathing are recurrent for you, just do exercises in the 5 minutes preceding your talk. Breath in deeply and breath out slowly. This will decrease your anxiety and will prepare your breathing for the talk. You can do it while seating in the audience or even while speaking with others.

Commit physically: avoid distractions

Personally, I have a routine. Before a talk, I remove everything from my pockets, or even the badge, anything superfluous. After a few talks delivered with my pockets inside-out dangling from my trousers, I also double-check that I am generally presentable! So, on stage or seating in front of a panel, I have no distractions from the badge hitting the microphone, the phone vibrating, the keys stuck in my thigh. Well, the phone: switch it off well in advance of your talk and dump everything in you bag.

Commit mentally: have fun

Those were a few suggestions, and more or different tricks will work for you, to ensure your physical state will be ready to support the potential stress you might experience while speaking in public. Of course, your state of mind will play an equally important role. Perhaps, I should advise to not care, to convince yourself that the event you are preparing does not matter. This is probably key, more in general, to achieve the resilience necessary in the academic world. For me that does not work very well, as I tend to be heavily invested in everything I do. So, what it works for me is to repeat myself I need to have fun speaking about science, my work, or the work of others – otherwise is really not worth. A bit of self-couching targeted to focus your mood towards excitement, how great can be to speak or debate science.

I did receive my dose of criticisms in my career, but let me tell you which is one of the best compliment I ever got. Do you remember the talk I was freaking out during my PhD? Well, after my talk, which might not have been even an excellent one, I overheard the head of a department advising two junior PIs to speak with the energy and enthusiasm I was speaking with. I guess you should remind yourself of how exciting the work you do is and if you disagree with this, change job or lie to yourself for a couple of hours.

Commit mentally: focus

You would not run the athletics world final 100m, physically unprepared and with no excitement. You would also not run it thinking about random stuff or worrying not to win it. Watch athletes on their blocks, the intensity of their eyes, the deep focus they concentrate on the start gun and those few seconds after. Focusing might take a fraction of a second if you were a natural or simply experienced. Also, keep your focus during the talk, try to nurture that unconscious little voice that can warn you everytime you are going off-track.

The top right-hand corner syndrome (TRiHCS) is a risky issue in our business. TRiHCS happen when your mind wonders off, but you keep speaking. TRiHCS happen when you zone out and speak for 2 minutes about an irrelevant detail being fixated on a corner of a room, while you are not engaging with the audience and perhaps even with the main topic of the talk. If you get TRiHCSed, your timing and narrative will derail. But, do not worry, if you notice it in time, you can easily recover.

OK, ok… TRiHCS? I just made this up, but I promise you, it is something that does happen!

Look after yourself…

Pay attention to yourself. It is easy to get anxiety compromise your health in the long term, or your performance in the short term. In an ideal world, you can sleep, eat, drink, meditate as a Yogi. In the real world, assaulted by too many things to do, it is likely you will experience periods of stress and long hours. However, you will have to know your limits and try to stay far from the edge and arrive to an event in good physical and mental conditions. Your institution and funders will offer you a provision of well-being courses, advice and activities. However, your institution and funders will implicitly ask you to neglect completely their own advice and deliver huge returns for them at any cost (for you). Like for any job, the day will come that you cannot run any longer over the edge. Then, manage anxiety, either it is just for public speaking, or for anything else… embrace it, as in ‘do not ignore it’, ‘do not fight it’ as it fights back, but manage it and if you can’t, ask for help.

Look after yourself… plan your cool-off stage

I did some crazy things aiming to present data still warm from the microscope (yes, it is a thing if you use high power lasers), consciously cutting sleeping times down (within reason) and working over the edge. Even if you do not, but public speaking really takes a toll on you, look after yourself after the main event. You need to consider two phases. One, which might be short or very long, depending on the event, is the immediate aftermath. I used to be a runner, and I used to give everything until the end of the race, which made it very likely for me to fall on the ground exhausted after the line… but you learn to immediately stand-up, walk, then do a run at slow space and hydrate.

Somehow, after a peak of stress you need to do something similar, often quietly and in public. This may have to happen in a few seconds before taking further questions. So, regain mental and physical composure, re-gather your focus and energy, again consider drinking water or a juice.  You will need this, particularly, in a day-long event full of meetings. It can really take just one minute, but if you do not do it, you might crash and underperform in the aftermath of a public speaking event. Do not underestimate the task you will have to follow after the main event and the energy you will need for them.

Then, at last, all is over. Really look after yourself because if the event you prepared took really a lot of energy from you, there might be consequences. You will discover what is best for you, if to completely relax and instruct yourself, or to simply take it easy for a few hours or a few days.

Conclusions

Keep in mind that what I have written here it is not an expert-opinion, but a personal experience. My suggestion to embrace your public speaking anxiety comes from trying to advise junior colleagues and realizing I did not wish to give the same suggestion a GP once gave to me: ‘you should avoid stress’. This is the wrong suggestion, in my opinion, as most of us, certainly in the ultra-competitive academic world, will have to manage plenty of stressful situation. Thus, the keyword is ‘manage’ not ‘avoid’, be the master or mistress of your stress-responses and, yes, avoid only those things that might push you too far beyond what you can manage. So, embrace your public anxiety speaking, mould your response to it in time and you will eventually grow out of it, or if not, at least you will manage.

Of course, whatever I described here is not something I usually think about, even during big talks. I made an effort to catalogue the various ‘tricks’ I – sometimes unconsciously -matured in 15 years of presenting scientific work in public. But recently, I had noticed that – either as a natural predisposition or by training – delivering a talk is more than just speaking in public. It is a process that requires physical and psychological strengths, like an actor preparing for a play or an athlete for a race. Scientists, noticing it or not, need to nurture these strengths, even not for their audience, but at least for looking after their health.

[TALK] Goldilocks and the two ERKs; signalling in the ‘sweet spot’ underpins resistance to ERK pathway inhibitors

Friday 14/09 at 14.30 | Dr. Simon Cook (Signalling Laboratory, The Babraham Institute) will present the following talk, at the Clifford Allbutt Lecture Theatre, Clifford Allbutt Building (former LMB building). All welcome to attend.

 Goldilocks and the two ERKs; signalling in the ‘sweet spot’ underpins resistance to ERK pathway inhibitors

Simon Cook, Signalling Laboratory, The Babraham Institute

Tumour cells with BRAF or RAS mutations are ‘addicted’ to ERK1/2 signalling for proliferation and RAFi and/or MEKi are now approved for use in the clinic.  However, despite some striking clinical responses, resistance emerges within 9-12 months resulting in disease progression. Acquired resistance to MEKi often occurs through amplification of BRAFV600E or KRASG13D which act to reinstate ERK1/2 signalling.

Here we show that BRAFV600E amplification and MEKi resistance are fully reversible following drug withdrawal.  Resistant cells with BRAFV600E amplification become addicted to MEKi to clamp ERK1/2 signalling at a level optimal for cell survival and proliferation (2-3% of total ERK1/2 active, quantified by mass spectrometry).  This is seen in cell culture and in vivo where growth of resistant cells with BRAFV600E amplification as tumour xenografts is inhibited in mice that do not receive MEKi.  ERK1/2 hyperactivation (~20% active) following MEKi withdrawal drives expression of the cyclin-dependent kinase inhibitor (CDKI) p57KIP2, which promotes G1 cell cycle arrest and senescence, or expression of NOXA and cell death; these ‘terminal’ responses select against those cells with amplified BRAFV600E.  ERK1/2-dependent p57KIP2 expression is required for loss of BRAFV600E amplification and determines the rate of reversal of MEKi resistance.  Thus, BRAFV600E amplification confers a fitness deficit during drug withdrawal, providing a rationale for intermittent dosing (‘drug holidays’) to forestall resistance.

Remarkably, MEKi resistance driven by KRASG13D amplification is not reversible. ERK1/2 reactivation in the context of amplified KRASG13D does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to chemotherapy agents, arguing strongly against the use of ‘drug holidays’ in cases of resistance to MEKi driven by KRASG13D amplification.