Category Archives: Pathological Cells and Processes

The Tumor Microenvironment “Big Tent” series continues (part 4)

 

The Tumor Microenvironment (TME) series to date is assembled here http://www.sugarconebiotech.com/?s=big+tent containing parts 1-3

I’m happy to point you to the most recent content, posted on Slideshare: http://www.slideshare.net/PaulDRennert/im-vacs-2015-rennert-v2

In this deck I review the challenges of the TME particularly with reference to Pancreatic and Ovarian cancers. A few targets are shown below.

Feedback most welcome.

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“Combination Cancer Immunotherapy and New Immunomodulatory Targets” published in Nature Reviews Drug Discovery

Part of the Article Series from Nature Reviews Drug Discovery, our paper hit the press today

Combination cancer immunotherapy and new immunomodulatory targets. Nature Reviews Drug Discovery 14, 561–584. 2015.  doi:10.1038/nrd4591

by Kathleen Mahoney, Paul Rennert, Gordon Freeman.

a prepublication version is available here: nrd4591 (1)

Immune Checkpoint Conference Interview (PDF)

Hit the link to see the full text (PDF) of Paul Rennert’s interview by Fiona Mistri, representing ICI15. The Immune Checkpoint Conference is being held next month in Boston.

Paul Rennert SugarCone Biotech LLC

The Big Tent: Halozyme is Targeting the Tumor Microenvironment, part 3 of an occasional series.

Many drug development programs claim to be truly unique and novel. It’s a mixed message really – complete novelty implies (or ensures) a high level of risk. It’s a bit difficult to attract early investment to such programs and maintain investor interest going forward. When we work with companies raising money, or are raising money ourselves, we are constantly trying to minimize risks, plural, as risks represent diverse aspects of a program or company: technology risk, biology risk, clinical risk, commercial risk, to highlight just a few. Companies that can move novel programs forward while derisking them in multiple areas certainly warrant our attention – for the scientific thesis and the investment thesis. We recently wrote about Innate Pharma, a company with first-in-class programs targeting NK cell immune checkpoint pathways (link 1). This is a good example of a company that has shed biology and clinical risks as the partnership with Bristol-Myers Squibb (BMS) continues to grow. The entire second tier of antibody-drug conjugate linker/payload companies (Redwood, Igenica, Mersana, Catalent and many others) will remain technology risk-heavy until each individual company either secures partnerships that eventually move ADCs into the clinic, or get their themselves. We could go on and on.

A few weeks ago I asked for companies and programs targeting the tumor microenvironment. Among the responses I got these:

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william gerber HA

Halozyme (Nasdaq: HALO) has a lead program that is very novel and I think scientifically is very interesting and has understood biological risk. We’ll talk about other risk elements in a bit, but science first. PEGPH20 is a pegylated version of the company’s approved recombinant human hyaluronidase (rHuPH20; brand name Hylenex). Hylenex is licensed to several partners, and provides a steady income stream from royalties. Hyaluronidase catalyzes the random hydrolysis of 1,4-linkages between 2-acetamido-2-deoxy-b-D-glucose and D-glucose residues in hyaluronan (HA), a constituent of the ECM. Hyaluronidase increases tissue permeability and as used locally (sc) to improve drug distribution. In the systemic tumor setting we have the interesting hypothesis that some tumor types use HA to create a cell impermeable “wall” around tumor cells or the tumor mass. The best-characterized tumor in this sense is pancreatic cancer, which is encased in an ECM that resists penetration by therapeutics and cells.

HALO is running a Phase 1/2 clinical program in PEGPH20 in patients with previously untreated metastatic pancreatic cancer. A completed Phase 1 clinical trial assessed the safety and tolerability of PEGPH20 treatment in patients with solid tumor malignancies refractory to prior therapies. A Phase 2 trial, built off a Phase 1b run-in, is underway in metastatic pancreatic cancer. The cohorts are standard of care (gemcitabine) with PEGPH20 or with placebo. An on-target toxicity (muscle spasm/pain) was addressed in a trial in which patients were pre-dosed with dexamethasone. At ASCO 2013, HALO presented data from the Phase 1b clinical study of PEGPH20 in combination with gemcitabine for the treatment of patients (n=28, 24 evaluable) with previously untreated stage IV metastatic pancreatic ductal adenocarcinoma (link 2). Patients received doses of PEGPH20 (1.0, 1.6 and 3.0 µg/kg) twice weekly for four weeks, then weekly thereafter, in combination with gemcitabine, IV. The RECIST 1.1 ORR (overall response rate = complete response (CR) + partial response (PR)) was 42% percent at the two higher doses. Subsequent exploratory analyses suggested better progression free survival (PFS) and overall survival (OS) in patients with high levels of tumor HA compared to patients with low levels of tumor HA. This has led the company to embark on the development of a companion diagnostic to enable pre-selection of patients.

Other clinical studies include a Phase 2 multicenter, randomized clinical trial first-line therapy trial of PEGPH20 in patients with stage IV metastatic pancreatic cancer. Patients were randomized to gemcitabine plus nab-paclitaxel with or without PEGPH20. The primary endpoint is PFS. SWOG has sponsored a Phase 1b/2 randomized clinical trial of PEGPH20 in combination with modified FOLFIRINOX chemotherapy compared to mFOLFIRINOX treatment alone in patients with metastatic pancreatic adenocarcinoma. MSKCC is sponsoring a trial +/- cetuximab. A full trial list is shown here:

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In October (2014) the FDA granted Orphan Drug designation for PEGPH20 for the treatment of pancreatic cancer. OK, so what do we see here? The therapeutic hypothesis is compelling, that disassembling the tumor-shielding ECM will be helpful (see link 3). Would this work as monotherapy? Perhaps, but that is not being tested, since keeping standard of care (SOC) on-board is important for these patients. But if we consider the impact of a disrupted architecture, I think we would argue that monotherapy, or at least interesting combination therapies, could be considered. The mechanisms of action are complex and include physical disruption of the tumor microarchitecture, disruption of aberrant circulation and interstitial pressure in the tumor, disruption of zones of hypoxia, and other effects. Look at this figure from the preclinical study (pancreatic cancer, mouse model):

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Panel A shows the dosing regimen (with gemicitabine), B shows the impact on pressure within the tumor and C v D shows representative tumors from the control and treated animals. Another recent paper discusses the vascular effects in detail (link 4). With the obvious leakage and loss of tissue integrity it makes sense to argue for combination with chemotherapy or antibody therapy, as in the cetuximab combo trial show above, from the MSKCC. One might also postulate that the collapse in pressure and increased access to the interstitial space might allow better penetrance by lymphocytes, allowing consideration of immune checkpoint combinations. But lets look closer:

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I left the figure legend in place so I don’t have to repeat the details, which show a reduction in smooth muscle actin (A v B) and collagen (D v E). Basically this figure suggests that the structural elements of the tumor microenvironment have collapsed. Given the impact on ECM components, I would predict that  you would see adverse impact on myeloid cell populations, inducing the TAM and MDSC populations discussed earlier (another link). I’d have loved to see a panel with PEGPH20 alone as I’ll bet you would see some impact with the monotherapy.

So if we go back to our three-legged stool model…

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… now we are dead-on the microenvironment piece, and perhaps an obvious complement to the other 2 legs.

All well and good but the proof is in the clinic, and so is the risk. We talked about diverse risks earlier – here we have clinical risk (efficacy/toxicity) and commercial risk (is it good enough). HALO is presenting at the ASCO GI meeting with abstracts to come out from under embargo on January 12, 2015. The abstracts will include an update on the clinical trial NCT01453153, phase 1/2 +/- gemcitabine in metastatic pancreatic cancer. Presentation of median OS data is rumored (but n.b. I’ve not confirmed with the company). I’m excited by the prospects here, and hope we see some nice results…

… because the science makes sense.

stay tuned.

The Big Tent: Tumor Microenvironment Targets Heat Up – part 2 of an occasional series

I recently asked folks for their favorite hot targets in the tumor microenvironment space. Among a flurry of responses I got these two related answers:

mcbio CSF-1R

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These responses from @mcbio316 and @Festivus159 were very timely, given what happened 4 days later (and a big shout-out to mcbio, whose post had preceded this):

Bristol-Myers Squibb and Five Prime Therapeutics Announce Exclusive Clinical Collaboration to Evaluate the Combination of Investigational Immunotherapies Opdivo (nivolumab) and FPA008 in Six Tumor Types

Five Prime Therapeutics, Inc. November 24, 2014 8:59 AM GlobeNewswire

  • NEW YORK and SOUTH SAN FRANCISCO, Calif., Nov. 24, 2014 (GLOBE NEWSWIRE) – Bristol-Myers Squibb Company (BMY) and Five Prime Therapeutics, Inc. (FPRX) today announced that they have entered into an exclusive clinical collaboration agreement to evaluate the safety, tolerability and preliminary efficacy of combining Opdivo (nivolumab), Bristol-Myers Squibb’s investigational PD-1 (programmed death-1) immune checkpoint inhibitor, with FPA008, Five Prime’s monoclonal antibody that inhibits colony stimulating factor-1 receptor (CSF1R). The Phase 1a/1b study will evaluate the combination of Opdivo and FPA008 as a potential treatment option for patients with non-small cell lung cancer (NSCLC), melanoma, head and neck cancer, pancreatic cancer, colorectal cancer and malignant glioma. Bristol-Myers Squibb has proposed the name Opdivo, which, if approved by health authorities, will serve as the trademark for nivolumab.

So BMS will immediately move FPA008, but all measures an early stage and largely unproven therapeutic, into combination therapy trials with nivolumab for the treatment of solid tumors. Not to be outdone, Roche has already positioned it’s CSF1R targeted therapeutic, as noted by @jq1234t:

 JQ screen shot

There are a number of interesting questions to answer here: What does CSF1R do, why is it so interesting, how does it impact the tumor microenvironment, how are these trials being done and (a favorite of mine), who else has assets in development?

CSF1R is the receptor for macrophage-colony-stimulating factor (aka M-CSF or CSF-1). The receptor is a control node for macrophage differentiation. CSF1R also serves as a receptor for the monocyte survival factor IL-34. Although the ultimate outcome depends on many factors, signaling through CSF1R is necessary for myeloid lineage precursor cell differentiation into macrophages, and it is this feature that interests us in the tumor microenvironment setting. We cannot gloss over the fact that this is a pleiotropic and complex biological system but it is safe to say that by the time we are confronted by an immunosuppressed tumor (as in the case of combo therapy with anti-PD-1/PD-L1 therapeutics), our pathway focus is on tumor associated macrophages (TAM), their impact on the tumor microenvironment and their susceptibility to CSF1R-targeted therapy.

Roche poached this figure that I’m now borrowing (with fair reference to Chen and Mellman, 2013).

Roche version Cancer Immunity Cycle

In the original figure (see Immunity 39: http://dx.doi.org/10.1016/j.immuni.2013.07.012 – an open access article), Chen and Mellman placed the PD-1 pathway inhibitors with a variety of microenvironmental modulators (IDO1, Arginase, TGFb) that together prevent, in distinct ways, cancer cell death. The Roche version of the figure, reproduced above, has been modified to include CSF1R among other targets in the “killing cancer cells” category.

Broad strokes, what does this mean? TAM, the tumor associated macrophages mentioned above, are dependent on CSF1R signaling. TAM are myeloid lineage-derived cells that are co-opted by the resident tumor as part of it’s microenvironmental support system. TAM are potently angiogenic, remodel the stroma (extracellular matrix and related components) and are immunosuppressive. Among the plethora of critical factors produced by TAM we find the hypoxia response proteins and growth factors that drive angiogenesis, tissue remodeling and immunosuppression, i.e. HIF2a, MMP-9, EGF, VEGF and TGFbeta, cytokines that can maintain this response in a chronic state (IL-10, IL-4) and chemokines that attract myeloid cells and regulatory T cells (CCL22, CCXL8). The TAM population can be directly regulated by tumor cell secretion of CSF-1, thus the importance of the CSF1R target. Multiple labs have produced preclinical data showing that anti-CSF1R antibody therapy can rapidly and effectively deplete tumors of the TAM population, and that this depletion has an impact on tumor growth and survival.

Clinical development to date is scattered. The FPRX program began with a Phase 1 trial in healthy volunteers and rheumatoid arthritis patients (NCT01962337) reflecting the role of diverse macrophage populations in inflammation and autoimmunity. Indeed the FPRX website states “we are currently conducting nonclinical research in areas such as idiopathic pulmonary fibrosis, lupus nephritis and other inflammatory disorders to identify a second target indication by the end of 2014″ although this may be trumped by the BMS deal. That trial reported safety and pharmacodynamic endpoints at AACR earlier this year. FPA008 was well-tolerated at all dose levels tested and the drug impacted inflammatory macrophage numbers and, interestingly, bone turnover (this latter effect due to the control of osteoclast differentiation by CSF1R, an important feature in bone metastasis settings perhaps).

In contrast Roche has been testing it’s antibody in a rare disease (a form of giant cell tumor) that is caused by a  t(1;2) translocation resulting in fusion of COL6A3 and M-CSF genes encoding for CSF1. The tumor is characterized by CSF1R+ cells. Roche reported that RG7155 had the following activities (Reis et al. 2014. Cell 25: 846–859):

- Anti-CSF-1R antibody depletes tumor-associated macrophages in cancer patients

- CSF-1R inhibitor shows clinical activity in diffuse-type giant cell tumor patients

- CSF-1R signaling inhibition increases lymphocyte infiltration in cancer patients

That last highlight referring to an effect on immunosuppression and refers to a relative increase of CD8+ T cells versus CD4+ FoxP3+ T regulatory cells, thus feeding the enthusiasm for combination therapy with anti-PD-1/PD-L1 therapeutics. More data is available in their ASCO abstract (http://meetinglibrary.asco.org/content/131522-144).

Other clinical stage antibodies include IMC-CS4 from Eli Lilly, in Phase 1 for advanced solid tumors (NCT01346358), ARRY-282 from Array BioPharma and Celgene, which had completed a Phase 1 trial in advanced solid tumors (NCT01316822) before the program was terminated, AMG 820 from Amgen with a completed Phase 1 study in advanced malignancies (NCT01444404), and others. Preclinical programs are visible at many small companies, both private and public, and include small molecule inhibitors of the receptor, e.g the Ambit and Plexxikon programs.

While the enthusiasm seems warranted by the preclinical modeling data and the (to date) apparent tolerability of the antibody therapies, I did receive this one note of caution from @Boston_Biotech:

Bos Biotech screenshot

Nuances, indeed. It is important to consider a few possible issues. First, blockade of CSF1R in mice led to the pronounced and sustained upregulation of CSF-1, and drug doses had to be kept high in order to “drug-through” this level of ligand to block the receptor. Rebound activity at trough or upon drug cessation could be a big problem, as has been described for other systems, including CCL2 blockade in breast cancer models (leading to abundant metastasis). Sticking with breast cancer, it has been reported that blocking CSF1R leads to upregulation of GM-CSF signaling, changing the composition perhaps (but not the stability) of the tumor microenvironment. Finally, as always, we cannot yet see what efficacy drugs will have as monotherapies (its too early) while we race ahead to combo therapies. While its all hands on deck to get these assets into patients, they won’t all work and certainly can’t be sure they will do no harm. However, that said, I think targeting components of the tumor microenvironment, including TAM, is our next best step forward, and I certainly will enjoy watching the data unfold.

next time … what wraps pancreatic cancer up so tight that you can’t treat it until it explodes in a deadly metastasis fireball?

cool stuff.

Hif, Hif, Hif, Hike!

Football season. Except is was 85 degrees here in Massachusetts today and felt more like mid-July. Thankfully there is “fallball” (fall softball season) so we got to enjoy that instead.

We got a good look at the convergence of immune and pathogenic pathways in this week’s issues of Science and Nature. Two papers in Science identify metabolic adjustments made by monocytes and macrophages that may support innate immune memory. The same pathway is hijacked by some tumors to redirect macrophage activity, as described in a very nice Nature paper.

Cheng et al from the Netea lab in The Netherlands used a b-glycan derived from the pathogenic fungus Candida albicans to “educate” monocytes, mimicking an infection event (Cheng et al). C. albicans b-glycan, a carbohydrate moiety, binds the dectin-1 receptor on monocytes, macrophages and other innate immune cells and induces cell activation. This activation response included changes in the epigenetic profile of the cells. The epigenetic signature suggests that monocytes “trained” by exposure to b-glycan alter their metabolic status, in particular by elevating aerobic glycolysis with increased glucose consumption. Key glycolysis enzymes such as hexokinase and pyruvate kinase were epigenetically upregulated, supporting the shift to glycolysis. Aerobic glycolysis produces lactic acid and increased lactate production was also observed: these b-glycan activated monocytes have really committed to this metabolic state.

This metabolic shift was mediated by signaling from dectin-1 to AKT and mTOR. This signaling pathway is responsible for many cellular responses, including induction of HIF-1α (hypoxia-inducible factor–1α). In turn, HIF-1α-dependent signals turn on many genes needed to adapt to the metabolic shift. This is a common tactic in hypoxic conditions for example. Blockade of any steps in the pathway abrogated the metabolic shift and prevented “trained immunity”. The role of epigenetic components in induction of the metabolic shift in monocytes was demonstrated using the epigenetic inhibitors methylthioadenosine, a methyltransferase inhibitor, and givinostat, a class I/II histone deacetylase (HDAC) inhibitor.

A second paper from the same group dives deeper into the monocyte to macrophage differentiation program (Saeed et al). Short-term culture of monocytes with LPS (a TLR4 agonist) or b-glycan yielded distinct macrophage populations. Serum culture (mimicking the homeostatic state) yielded yet a 3rd type. This paper is a technical grind so have at it if you want all the complex details. I was interested in the conclusions. As in the b-glycan study referenced above, LPS and serum culture induced distinct epigenetic signatures. Genome-wide mapping of histone modifications identified epigenetically marked clusters – that is, reactive regions of the genome. Within these clusters we would expect to find transcription regulatory regions, and indeed four such clusters were differentially modulated when monocytes were exposed to LPS or b-glucan. Targets within these clusters include G protein–coupled receptors, protein kinases, and additional epigenetic enzymes. The authors therefore affirm the “trained immunity” state identified in the first paper and now elucidate a macrophage “exhaustion” phenotype induced by short term exposure to LPS. By my reading of the paper it appears both of these induced phenotypes are extensions of the M-CSF/serum induced homeostatic differentiation profile. This makes sense, as monocytes are recruited from circulation so they can differentiate into macrophages at sites of inflammation, a process that optimally requires M-CSF.

In the first paper the production of lactic acid and lactate was noted as a consequence of differentiation to the “trained”, glycolysis-driven phenotype. Turning now to a paper in Nature from Medzhitov and colleagues at Yale, we find ourselves confronting a chicken and egg story (Colegio et al). In this study the crosstalk of tumor-resident macrophages and “client” tumor cells was examined. The premise is that tumor-associated macrophages (TAMs) perform key homeostatic functions that support tumor growth and survival. In this case it appears that the tumor microenvironment subverts macrophage function via production of lactic acid. There are important differences in the study designs – the papers published in Science use short-term culture techniques while the Nature paper relies on in vivo tumor/macrophage development in syngeneic mouse models – but with this caveat in mind the convergence of pathway data is striking. TAMs sorted from implanted lung (LLC) or melanoma (B16-F1) tumors expressed high levels of VEGF and arginase 1 (Arg1) mRNA, accounting for nearly all of the expression of these proteins in tumor samples. Strikingly, tumors induced macrophage expression of VEGF via stabilization of HIF1a in a manner that did not require hypoxia. This is interesting as it identifies a pathway by which tumor cells can stimulate angiogenesis (blood vessel formation) via VEGF and Arg1 prior to a hypoxic challenge. The soluble tumor cell effector capable of turning on this pathway was identified as … lactate. Here it is worth quoting from the paper:

“Warburg observed that cancer cells preferentially perform aerobic glycolysis: that is, they convert most glucose molecules into lactate regardless of the amount of oxygen present. Furthermore, the eponymous Warburg effect is also observed in most cells undergoing rapid proliferation. It has been hypothesized that aerobic glycolysis is conducive to cell proliferation because, despite the consequent reduction in ATP production, aerobic glycolysis produces metabolic precursors, such as lactate, for biosynthetic pathways, and these precursors may be the limiting factor during rapid cell proliferation”

The suggestion here is that tumor cells are going a step further in order to ensure that their supportive microenvironment, which includes TAMs, step in line. Lactate is taken up by TAMs via specific cell surface receptors (the monocarboxylate transporters) and the effect is potentiated by acidic pH (from all the lactic acid) and perhaps requires other mediators such as M-CSF. Once all is said and done the TAMs are surviving and thriving using the same machinery as the tumor cells.

From the drug development perspective it is probably worth asking whether AKT and mTOR inhibitors impact TAM activity in the tumor microenvironment (perhaps someone already has). Conversely, one might speculate on the impact of such inhibitors of macrophage responses to infection. More selectively, I suspect there is a clever way of targeting the epigenetic responses to derail the TAM phenotype and disrupt the tumor-supportive microenvironment while either simultaneously targeting the tumor, as in a combination therapy setting with a therapeutic that targets tumor biology directly. Also, in the era of immune checkpoint therapeutics I wonder if there isn’t some signal to “wake-up” these “trained” macrophages and have them turn on their clients – the tumor cells.

A few other questions:

How is the macrophage glycolysis pathway maintained once initiated by exposure to tumor derived lactate? There must be a feedback mechanism, perhaps similar to the one used by “trained” macrophages?

Do the HIF2-dependent tumors (some renal cell carcinomas for example) also hijack resident TAMs in the same manner, or different?

The tumor microenvironment includes tumor-associated fibroblasts – are these also impacted by exposure to lactic acid?

If there is intimate cross-talk between the macrophage and it’s client (a tumor cell) then disabling that conversation at the level of the macrophage (and other stromal cells) should be therapeutic – or will the tumor (in this case) simply adapt? Remember that in this setting the epigenetic changes are not necessarily addictive (oncogenic).

interesting stuff to consider in this new era of combination therapies….

stay tuned

Creating a New Therapeutic Focus

In 2012 we were engaged by a large local biotech company to evaluate a new therapeutic area. This effort was driven by the desire of the client to move aggressively into a new suite of diseases. We began by doing a deep dive into the client’s existing portfolio in order to identify assets already in development that could be directed to novel diseases. Concurrently we began a comprehensive review of preclinical and clinical stage assets available for partnering or in-licensing. Finally we engaged in pathological pathway analysis to identify novel targets for discovery programs. This effort, initiated and completed within two quarters, led to the eventual acquisition of a private company and its Phase 2 clinical stage assets, for nearly 100MM $USD. Based on our analyses the client also started several new discovery and preclinical development programs to complement the clinical stage acquisition.

Cautionary Tales from Human Microbiome Frontier

The concept of symbiotic microbiomes (yes, plural) influencing our health seems now, in hindsight, to be obvious, and the fact that the science has caught up to the folk medicine has all sorts of people buzzing. Some of the buzz is well informed (see below), some not, but all in all we are making progress understanding a few of the ways in which our vast mucosal environment interacts with the outside world. At the same time its fair to say that we know very little yet, and have a long way to go. Some recent findings drive this point home.

We can think of the frontier mentioned in the title in two ways. One, maybe obvious, is to think about the frontier of science, as this is where we find ourselves as the technology to do the some of this work was not widely available until recently (e.g. affordable deep sequencing). More subtly, we can think of the mucosal environments – oral, pulmonary, digestive, excretory, reproductive – as frontier environments where self interacts with non-self in an exploratory manner, that is, not confrontational a priori. There is a lot at stake: pathogen recognition and defense, nutrient uptake, metabolic regulation, waste disposal, on and on.

It makes sense that there are tightly controlled and very complex rules of engagement. The new findings I want to review touch on some of these rules and suggest layers of control and organization that we really don’t understand yet. Secondarily, we can study these systems with an eye on drug discovery.

Back to back papers in the December 16/26 double issue of Nature identify a critical pathway for the development of regulatory T cells (Tregs) in the gut. Data from the Ohno lab in Japan and the Rudensky lab in NYC paint broadly similar stories of the role of the specific commensal bacteria in fostering Tregs (see references 1 and 2, below). Both papers show that the fatty acid butyrate stimulates the development of Tregs. This in itself is not a new finding. Butyrate is a major energy source in mammalian metabolism and not surprisingly it’s production is driven by commensal bacteria, notably the abundant Clostridia class of bacteria (some species within Clostridia are pathogenic, but that’s a different story). Again, it’s not particularly surprising that one of the most abundant mammalian commensals gives off good vibes in the form of fatty acids that support a quiet immune system. The papers differ in some curious ways, in particular, the Ohno paper states that the induction of Tregs was limited to the gut, while the Rudensky papers highlight Treg production in the lymph nodes and spleen, but not the colon. Regardless, the reason these papers made it into Nature is that they identify the mechanism by which butyrate induces Treg differentiation, and this is by inhibiting a histone deacetylase (HDAC IIa) thereby allowing for the specific acetylation (and therefore activation) of DNA elements that support Treg differentiation, notably at the FoxP3 promoter and enhancer.

Cool.

But before we all run out and start swallowing a bunch of butyrate capsules and subject ourselves to butyrate enemas (yes, both are available), lets be clear about what these papers are saying and what they are not saying. First, we are dealing here with inbred mouse strains on carefully defined diets. Translation of the results to outbred humans on diverse diets is not so straightforward. That said, the results support eating a high fiber diet, which will yield plenty of butyrate and related fatty acids. Second, the papers agree on one thing very specifically, which is that the generation of Tregs in the gut is a local phenomena, specific to the colon (large intestine, south of the caecum). This makes sense of course, as that is where the Clostridia are cranking out the fatty acids. The application of these findings to colonic disease, notably Ulcerative Colitis, is worth exploring. But broadening the scope to include general health, well-being and immune serenity is not warranted – despite the pile on by the Supplements and Wellness Industries.

A very different story just came out in PNAS (reference 3), and this one concerns the response of different populations to a gut pathogen found in the gastric mucosa (lining of the stomach). The bacterium Helicobacter pylori is found in about half of the human population worldwide. H. pylori is a causative agent of gastric adenocarcinoma in a small percentage of the people who are infected, less than 1%, although hotspots are known. One such hotspot was studied by a team from Vanderbilt who found that the higher incidence of H. pylori induced precancerous inflammation correlated with the presence of a European strain of the bacterium infecting an Amerindian population in Columbia. In contrast, an African strain of H. pylori infecting the descendants of African slaves nearby did not cause inflammation and cancerous lesions. The investigators conclude that H. pylori is mainly pathogenic when it occurs in a population distinct from that with which it co-evolved. So, a fine line between commensal and pathogen is drawn.

Ok, one more.

The gut microbiome has been implicated in the development of Th17 effector T cells, at least in mice. This is interesting in light of where we started, with the generation of Treg cells, since in some ways Tregs and Th17s are the result of different developmental pathways that T cells take. Note that the first two studies reviewed were focused on extrathymic (in that case, colon-specific) Treg generation. Mice that are raised with no pathogens in their environment, including their food, which is irradiated, don’t develop very many Th17s as a percentage of the total T cell population. Since Th17 cells are associated with diseases (including rheumatoid arthritis (RA), psoriatic arthritis (PA), psoriasis, inflammatory bowel disease) it seems reasonable to ask whether a Th17 inducing microbiota is linked to any particular disease. Littman’s lab at the Rockefeller in NY has done exactly that (reference 4). Newly diagnosed RA patients were found to carry the intestinal bacterium Prevotella copri at much higher levels (75%) than PA patients (37%) or healthy control patients (21%). This association of a specific pathogen with an autoimmune/chronic inflammatory disease is very striking. When mice were infected with a rodent-compatible strain of P. copri they developed pronounced intestinal inflammation, but not arthritis. Still, the intestinal inflammation was associated with the induction of Th17 cells, and so the hypothesis that this may underlie more systemic inflammation (e.g. RA) is still reasonable.

There are some problems with the story. The clinical development of IL-17 targeting drugs has shown that these do very well in PA and psoriasis, perhaps in inflammatory bowel disease, but they have failed to show sufficient benefit so far in RA. So at the level of drug discovery the link of an intestinal pathogen to Th17 T cells producing IL-17 and then to the disease, RA, seems to falter.

Thinking more broadly, the application of microbiome studies to drug development is in its infancy, and I think there is some reason for optimism as these studies become more sophisticated. The H. pylori and P. copri studies mentioned make it clear that many factors influence the response of a given population or individual to their microbioma. One interesting approach, the use of fecal transplantation to treat severe diarrhea and also Crohn’s disease, has made it into early clinical trials. Isolation of the critical components that reset the immune system in the local (inflammatory bowels diseases) and systemic (RA and other non-gut inflammatory diseases) settings is going to take significant time and effort, so we’ll have to stay tuned.

References
1) Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells, Nature, http://www.nature.com/nature/journal/v504/n7480/full/nature12721.html
2) Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation, Nature, http://www.nature.com/nature/journal/v504/n7480/full/nature12726.html
3) Human and Helicobacter pylori coevolution shapes the risk of gastric disease, PNAShttp://www.pnas.org/content/early/2014/01/08/1318093111
4) Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis, elife, http://elife.elifesciences.org/content/2/e01202

Cells underlying fibrosis: emerging themes

by Paul D. Rennert, 28 December 2013
A few weeks ago a paper describing the origin of cells responsible for dermal wound healing showed up in Nature, with a beautiful cover picture showing distinct cell lineages in skin. I actually missed the paper when it came out, so thanks to my friend Katherine Turner (ex-GI, ex-Biogen, now at Scholar Rock) for pointing it out.
The abstract is here, the full text requires a subscription. The paper is from Fiona Watt’s group at the Center for Stem Cells and Regenerative Medicine, King’s College, London.
The paper convincingly shows that the dermal layer of the skin is comprised of two distinct fibroblast lineages. Some context: the dermis is the layer of the skin that lies below the epidermis and includes the upper dermis (the dermal papilla) and the hypodermis (lower dermis). The major cells types residing in the dermis are fibroblasts, which along with the extracellular matrix they produce, form the connective tissue. Other prominent cells include macrophages and adipocytes (fat cells). There are also resident dendritic cells, Mast cells, T cells and other immune system cell types. Other structures include the hair follicles, sweat glands, oil glands, blood vessel endothelium, lymphatic vessels, and nerve endings.
Ok, so why is this paper interesting?
What the authors demonstrate is that the upper and lower dermis originate from distinct lineages of fibroblasts. This is shown using lineage tracing techniques and cell transfer assays. The lineage tracing assays required the use of multiple promoter/expression transgenic mice, and is elegantly and robustly done. The data show that late in mouse embryonic development (day 16.5) the dermis becomes fate-restricted: the Lrig1+ fibroblasts become the cells of the upper dermis and Dlk1+ cells give rise to the lower dermis. Here I’ve just picked a few of the markers they used and have grossly simplified the developmental story because I’m more interested in what they did next.
Having established that there are 2 different fibroblast lineages in the mouse dermis, the investigators asked a pretty simple question: what happens when you wound the skin in the adult mouse? Cellular wound-healing models have been widely used as in vitro models of fibroblast migration, ECM production and differentiation into myofibroblasts (so called because they express smooth muscle actin, a marker of myocytes/smooth muscle cells). Wound healing more broadly is a process of local repair and remodeling. “Deranged” or unregulated wound-healing is one model of fibrosis.
Skin or dermal fibrosis is a highly active area of study. Important fibrotic diseases of the skin include systemic sclerosis (scleroderma), amyloidosis, nephrogenic systemic fibrosis, mixed connective tissue disease, wound-healing fibrosis and others. Scleroderma is modeled in mouse by creating chemical insults on the skin, for example with bleomycin. Such models mimic the effect of chronic wounding, thus the connection to wound healing assays. It has been further demonstrated that many of the processes and pathways involved in dermal wound-healing are dysregulated in skin fibrosis, for example ECM production, TGFbeta production, and CTGF activity among many others.
While there is a good deal of consensus as to the mediators of the fibrotic process, the source of the activated cells that drive fibrosis has been controversial. This is a critical question to address, as effective anti-fibrotic therapies should target the pathogenic cell types. Without getting into immune cells, resident macrophages and Mast cells and the like, we can now at least carefully address this question regarding the source of activated fibroblasts. One model proposes that migrating fibrocytes traffic into wounded/fibrotic sites from the circulation. Another model, widely held, proposes that pericytes, which are mesenchymal cells that support blood vessels, somehow migrate away from endothelium and become myofibroblasts, thus supporting wound healing (and by extension contributing to fibrotic diseases). Finally, it has been proposed that myofibroblasts are derived from local (resident) fibroblasts that migrate into the wound and differentiate.
The Watt’s paper shows clearly that the Dlk1+ fibroblasts of the lower dermis migrate into the wound and that a proportion of these cells express smooth muscle actin and are therefore myofibroblasts. These cells produce collagen, the classic ECM protein that characterizes fibrosis, in this case simply deployed to help close the wound. After the wound is closed the epidermal layer is reformed and only then does the upper dermis reform.
These findings allow for the formulation of specific questions regarding the lower dermal fibroblasts:
- what signals activate and then shut down the lower dermal fibroblast to myofibroblast differentiation process?
- are these signals dysregulated in fibrosis?
- do these same cells deploy in response to fibrotic injury (e.g. with bleomycin)?
- do these cells interact with inflammatory cells, thought to contribute to the pathogenic fibrotic response?
- can we identify actionable targets on this cell type to prevent or decrease the fibrotic response?
Importantly this paper encourages us to ask whether similar resident fibroblast or other mesenchymal cell populations contribute to fibrotic disease in other organs such as the kidney, liver, heart and perhaps even lung. If so, the validity of the circulating fibrocyte and differentiating pericyte models might be reevaluated. Notably, linage tracking was used in support of a pericyte model of myofibroblast differentiation in a similar dermal/muscle injury model as detailed in a paper published in 2012 in Nature Medicine (abstract here). I suspect that squaring these studies will take a careful review of the markers used to identify different cell populations and a detailed look at the injury protocols. I’ll update as more info becomes available.
This is a classic example of the beautiful work that comes from an active and competitive research area and we can and look forward to further studies that will illuminate the processes of wound healing and fibrosis. Such work will certainly support and advance successful fibrosis drug development.
you may leave a comment or contact me:
rennertp@sugarconebiotech.com
on Twitter @PDRennert
cheers-
Paul

Conference Update: Targeting the Wnt Pathway in Oncology

October 29, 2013
At the ASCO and the AACR/NCI/EORTC meetings there was an avalanche of publicity about immunotherapies, combination therapies, mechanisms of tumor resistance and tumor genetics: all areas of intense importance and astounding progress. New therapeutic modalities, such as epigenetic regulation, also received great attention. Less noticed, but important I think, was the slow but steady progress being made toward the effective targeting of the Wnt pathway.
The Wnt pathway leading to oncogenic activation of β-catenin has been studied for decades but only now are we seeing effective means of antagonizing Wnt signaling. The importance of the Wnt pathway in oncogenesis was revealed in the course of investigation of tumor causing murine retroviruses during the 1980s and 1990s. Work in the Varmus lab and others led to the discovery that the Mouse Mammary Tumor Virus (MMTV) was oncogenic due to a proviral insertion that activated a gene called int1. Int1 was subsequently renamed Wnt1, based on homology of the protein to the Drosophila family of Wingless proteins (encoded by the Wg genes). For a beautiful review of the field see Nusse & VarmusEMBO J. 31: 2670-2684.
The Wnt proteins are secreted from cells as 350 – 400 amino acid lipid-modified glycoproteins. The lipid modifications are required for effective cell secretion and also for receptor binding. For example, the Porcupine protein, an O-acyltransferase, is required for palmitoylationthat allows efficient secretion of Wnt. All Wnt proteins (there are more than a dozen in mammals) bind to the Frizzled receptors, a large family of G-protein coupled signaling receptors (GPCRs). Frizzled is most often found in a cell surface complex with co-receptors, notably the low-density lipoprotein-related protein, LPR5/6. Binding to the receptor complex triggers complex and fascinating signaling cascades. Signaling is mediated by phosphorylation of the cytoplasmic protein Dishevelled (Dsh), that signals through several distinct activation domains. Critical to cell activation is Frizzled/LRP5/6-mediated displacement of the negative regulatory complex that includes the proteins Axin, APC, GSK3β and several others. This negative regulatory complex is called the “destruction complex” since its normal function is to degrade, via the ubquitination/proteosome pathway, the critical Wnt pathway signaling protein called β-catenin.
Accumulation of b-catenin leads to translocation from the cytoplasm into the nucleus, and interaction with the transcription factors TCF/Lef1 and the Creb-binding protein (CBP). Many genes are known to have TCF/Lef1 and CBP binding sites in their promoters and are therefore potential targets for Wnt signaling. Many of these genes in turn have been implicated in tumor genesis, growth and survival. Notable genes targeted by β-catenin signaling include c-myc, Cyclin D, c-jun, various growth factors, and many others. Both TCF-1 and Lef-1 are up-regulated in an autocrine manner, further propagating β-catenin-dependent signaling.
Evidence for the critical role of the Wnt pathway in tumor pathogenesis has come from genetic studies of the pathway’s different components. Accumulation of β-catenin has been observed in diverse cancers. Mutations associated with cancer include the loss-of-function mutations of APC in colorectal cancer that decrease the rate of β-catenin degradation.  Mutations have also been described in the β-catenin gene CTNNB1 and in the Axin gene (AXIN1), among others. The effect of the most common of these gene mutations is to prevent degradation of β-catenin. Other mechanisms of Wnt pathway regulation are described below in the context of drug development.
Critical advances in the understanding and targeting of the Wnt pathway in cancer were presented this year at the ASCO annual meeting in May and at the AACR-NCI-EORTC “Molecular Targets and Cancer Therapeutics” conference held last week in Boston. These advances specifically address the role of aberrant Wnt pathway signaling in the context of tumor cell proliferation and survival, and also in the emerging field of cancer stem cell biology. The potential of this pathway in cancer therapeutics is indicated by the appearance of pathway antagonists in biotech and pharma portfolios.  Examples are given below, and there are certainly additional efforts underway.
Several compounds have reached clinical trials, including both small molecules and biologic drugs. A leading therapeutic class is the Porcupine inhibitors, as exemplified by LGK974 from Novartis. Porcupine inhibitors reduce O-acyltransferase activity by Porcupine and thereby antagonize Wnt protein secretion. LGK974 has shown activity in preclinical tumor models and is currently in Phase 1/2 clinical trials in melanoma and breast cancer to establish dose and tolerability. Other porcupine inhibitors are in preclinical development and some of these are listed on the Wnt homepage (http://www.stanford.edu/group/nusselab/cgi-bin/wnt/smallmolecules). Preclinical data using a second porcupine inhibitor, C59, was reported at the AACR-NCI-EORTC meeting. Using this inhibitor, Wnt-dependent tumor growth was blocked in xenograft tumor models, without evidence of overt toxicity.
There are even more compounds in preclinical development that act by stabilizing the Axin protein, thereby maintaining the “destruction complex” and preventing β-catenin accumulation. One is XAV939 from Novartis, an antagonist of tankyrase (TRF1-interacting ankyrin-related ADP-ribose polymerase; TNK). TNK antagonists act by inhibiting the enzymatic activity of TNK1 and TNK2 that act to mediate Axin ubquitination and proteosomal degradation. Axin targeting is being pursued aggressively for two reasons: first Axin mutations are associated with increased levels of β-catenin in diverse cancers, including colorectal carcinomas, hepatocellular carcinomas, and medulloblastomas. Second, interesting work has suggested that Axin is the rate-limiting component of the “destruction complex” at least in some experimental systems. The Novartis compound XAV939 is one of numerous TNK1/2 inhibitors in preclinical development. Genentech has reported that its inhibitor (G007-LK) was active in models of colorectal cancer cells carrying APC loss-of-function mutations. This is a critical therapeutic profile if such inhibitors are to find wide utility. The Genentech program has been licensed to Odin for use in colorectal cancer. A recent paper presented structural models of the binding of G007-LK and a novel inhibitor WIK14 to tankyrase (Haikarainen et al. PLoS ONE 8: e65404). Programs from Kyowa Hakko Kirin and others are now visible in various publications, abstracts and patents.
Another small molecule, PRI-724, blocks the interaction of β-catenin with the transcription factor CBP to prevent pro-growth and pro-survival gene expression. PRI-724, developed by PRISM in collaboration with Eisai Pharmaceuticals, is in Phase 1 clinical trials in AML and advanced solid tumors. Inhibitors of T-NIK activity are also being advanced. T-NIK is an activating kinase for some TCF transcription factors, and appears to be required for colorectal cancer cell proliferation. Astex Pharmaceuticals (now owned by Otsuka) has an active preclinical program. Carna Biosciences presented characterization data on a tool compound at the AACR-NCI-EORTC meeting.
Anti-Frizzled receptor antibodies constitute a distinct class of Wnt pathway inhibitors. The most advanced of these, vantictumab (OMP-18R5) from OncoMed/Bayer. This antibody binds to five of the frizzled receptors, and is in Phase 1 clinical trials, with interim data reported at meetings this year. Patients with advanced, refractory solid tumors were treated with single-agent vantictumab at doses up to 15 mg/kg every three weeks. The investigators have stated that the 15 mg/kg dose maintained an efficacious exposure, based on rodent tumor models. Evidence of single-agent activity of vantictumab was noted in several neuroendocrine tumor patients. An interesting question is whether this effect on the tumors was on mechanism (i.e. due to inhibition of Wnt/Frizzled interaction) or due to effector function of the antibody, sufficient to induce cell killing at the site of antibody binding. At the AACR-NCI-EORTC meeting PD data was presented that was clearly on mechanism, based on the regulation of stem cell and differentiation genes expressed in tumor and hair follicles (sampled) and bone (inferred from blood samples). PD effects were noted at all doses examined. OncoMed and Bayer are also developing a Frizzled-Fc fusion protein, mimicking a normal means of regulation by secreted extracellular domain fragment of Frizzled proteins by cells.
The discussion of PD markers in response to Wnt pathway inhibition brings up several interesting issues. Effective inhibition of Wnt pathway signaling will potentially block normal stem cell renewal, most critically of the intestinal epithelial compartment. This single cell epidermal layer creates the mucosal barrier that maintains the sterility of the mucosal tissues lying adjacent to the lumen running from the mouth to the anus. Compromising the integrity of the mucosal epithelium can lead to toxicity ranging from gastrointestinal discomfort to more severe manifestations resulting in sepsis.  The turnover of gut epithelial cells is 4 days in human, meaning that any ablation of stem cell cycling would have an impact fairly quickly. Therefore maintaining a therapeutic window will be critical in the setting of Wnt antagonism.
An area of intense recent interest has been in the field of cancer stem cells, putatively acting not only as oncogenic progenitors but importantly as the source of resistant populations following conventional (chemo, radiation) or targeted (rational, immunotherapy) treatments. The alarming spread of highly aggressive treatment resistant cancer that occurs in many or most settings of solid tumor therapy speaks to the importance of the stem cell like properties of some cancer cells. The extent that stem cells from different tumor types are dependent on Wnt signaling has not yet been determined. The best data come from studies of colorectal cancer. Demonstration that gut stem cells and their oncogenic progeny are dependent on Wnt signaling was beautifully presented by Hans Clevers at the AACR-NCI-EORTC meeting last week (see his review at Cell 54: 274-284). Recent published data also suggest a critical role for the Wnt pathway in maintaining the complex signaling matrix required to support glioblastoma proliferation (PLoS Comput Biol. 9: e1002887).
Another interesting question with respect to Wnt pathway antagonism is where in the pathway to intervene. Upstream antagonists such as the anti-frizzled antibody or the porcupine inhibitors may not be effective in cases where the downstream components have been mutated. Therefore, such therapeutics may best be used in the context of a high β-catenin signature absent Axin or APC mutation. Downstream antagonists such as β-catenin-CBP antagonists may have potentially useful specificity but may not provide sufficient inhibition of the signaling cascade. In this context, tankyrase inhibitors appear to be sitting in the right spot. Finally, alternative pathways to β-catenin activation have been described although it is not understood yet to what extent these pathways are active in tumor biology in situ. These alternative pathways may not be targeted by current therapeutic approaches.
Regardless, the Wnt antagonism field has now grown to encompass diverse intervention points, and the first antagonists have entered clinical trials. Early signs of success, if they come, will no doubt continue to drive interest in this critical oncogenic pathway.