Category Archives: drug mechanism of action

Hif, Hif, Hif, Hike!

cancer, drug mechanism of action, Pathological Cells and Processes, TAMs, Uncategorized

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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

ANTICIPATING NEW THERAPEUTICS AND FORECASTING TREATMENT TRENDS FOR ACUTE MYELOID LEUKEMIA – PART 1

AbbVie, AML, Companies - stock symbols, drug mechanism of action, Flt3, HDAC, Jak2, Karyopharm, Merck, midostaurin, Novartis, Onyx Pharma, panobinostat, Pathways for Drug Development, plerixafor, Quizartinib, sorafenib, vorinostat

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 by Paul D Rennert, February 11, 2014

In looking at Acute Myeloid Leukemia (AML) we see a cancer field right on the cusp of change in clinical practice. Standard of care chemotherapy regimens and stem cell transplantation protocols have proven to be of limited utility, especially in older patients. However, potentially big advances in care are being made, with exciting news coming out regularly. As we move toward the spring Medical Conference season, we felt an overview of this rapidly evolving area of oncology would be timely.

AML is a rapidly growing cancer of myeloid lineage cells that proliferate in the bone marrow and interfere with normal hematopoiesis. AML typically arises in the context of defined genetic mutations. For example, translocations of chromosome 16 disrupt RUNX1 gene activity and are one of the several underlying causes of Core Binding Factor AML. CBF-AML). Since RUNX1 regulates the transcription of many genes, the effect of its disruption is complex. CBF-AML patients are generally responsive to chemotherapy initially, although up to half of these patients will relapse over time due to additional genetic mutations.

Mutation of the FLT3 protein is the most common genetic abnormality in AML, found in about 30% of patients. This is a genetic characteristic associated with poor prognosis. The most common FLT3 mutation, FLT3-ILD, is caused by an tandem duplication within the coding region of the gene. The resulting protein drives hyper-signaling and oncogenic cell responses. Mutations that change the active site of the protein, causing unregulated phosphorylation, have also been described. Mutations in the receptor tyrosine kinase c-Kit are also associated with oncogenic signaling in AML. Both of these pathways cause mutiple downstream effector pathways to be activated. The JAK2 mutations, commonly see in myelofibrosis and other myeloproliferative disorders, are rare in AML but when characterized can potentially be treated with Jak2 inhibitors.

According to a recent market research analysis                           (http://www.transparencymarketresearch.com/acute-myeloid-leukemia-therapeutics-market.html) a total of 62,226 new cases of Acute Myeloid Leukemia (AML) were recorded in 2010, with 95,000 predicted new cases for 2015 and nearly 130,000 predicted new cases in 2020. Note that as of February 2014 approved agents for AML remain limited to chemotherapeutics (http://www.cancer.gov/cancertopics/druginfo/leukemia#dal2). Despite the lack of new targeted drugs, the AML therapeutics market was nearly 240 MM USD in 2011. At the current rate of growth the AML market could reach over 700 MM USD by 2018. These numbers are based on the analysis of future AML drugs growing at a 17% compound annual growth rate from through 2018.

Numbers like these are continuing to drive intensive research into effective, novel therapies for AML. It only helps that in many cases such therapeutics find use in other hematopoietic diseases such as Chronic Myeloid Leukemia (CML) and in the B cell lymphomas, including Hodgkin’s Lymphoma and the non-Hodgkin’s Lymphomas (NHL).

 There is obvious unmet medical need for effective therapies in AML since this is a disease characterized by quick relapse after therapy with grim survival statistics. In some older patients, survival is as little as 1-1.5 years despite first and second line treatment regimens.

What’s exciting from the drug development and biotech investment perspectives is that the AML treatment landscape is advancing simultaneously across therapeutic modalities. This rapidly changing landscape give us a chance to look at targeted small molecule drugs, monoclonal antibodies (naked, bi-specific, radiolabelled, immunotherapeutic, ADC), targeted T cells and other novel technologies.

 We can then ask ourselves: who will the winners be in 5 years?

 A) Targeted small molecule drugs.

Lets just be clear upfront that the goal of these targeted therapies is to get patients who have relapsed, or are refractory to chemotherapy, to a complete response (CR) with minimal residual disease (MRD) so they can qualify for an allogeneic stem cell transplant (SCT). That’s a lot of acronyms but what this is really saying is that for most patients the goal is a modest one – we are not asking for a durable remission, at least not yet.

 A variety of established drugs are being tested in AML. Also, the identification of oncogenic mutations in FLT3 and cKIT has driven interest in developing new tyrosine kinases inhibitors (TKIs) for AML.

 Sorafenib (NexavarTM; Bayer and Onyx) is a dual targeting drug that blocks RAF signaling (and therefore the MEK>ERK signaling) and also the growth factor receptor tyrosine kinases VEGFR and PDGFR. The NCI is running a large phase 3 trial enrolling new onset pediatric AML patients (NCT01371981) with sorafenib being given in combination with various chemo regimens.  Bayer and Onyx are running several earlier phase AML trials. An interesting phase 1 trial in patients 18 or older combines sorafenib with plerixafor and G-CSF (NCT00943943). The idea here is to have the CXCR4 blocker (plerixafor) and the growth factor (G-CSF) flush tumor cells, and also tumor stem cells, from the bone marrow and lymph nodes so that they are more sensitive to sorafenib treatment. This trial is co-sponsored by Genzyme/Sanofi, which owns plerixafor.

Another interesting trial is the Phase 1/2 study of the combination of sorafenib, with vorinostat, and bortezomib (NCT01534260). Here we have a proteasome inhibitor and an HDAC inhibitor added to growth factor and signaling inhibition provided by sorafenib. This potent combination is being used in patients with a poor genetic risk profile, including FLT3-ILD positive tumors. This study is co-sponsored by Bayer/Onyx, Millennium/Takeda and Merck Sharp & Dohme Corp.

Bristol Myers Squibb is running an interesting trial (NCT01620216) in which AML and acute lymphocytic leukemia (ALL) patient samples are analyzed for sensitivity to drug treatment ex vivo, after a period on drug in the trial, as follows:

“An in vitro kinase inhibitor assay will be used to determine the sensitivity of primary leukemic cells to four kinase inhibitors/drugs:

Drug: Sunitinib, 50 milligrams (mg) qd, with or without food, for 4 weeks

Drug: Dasatinib, 100 mg qd…possible escalation to 140 mg qd for 28 days

Drug: Nilotinib, 400 mg twice daily for 28 days

Drug: Sorafenib, 400 mg (2 tablets) orally twice daily without food for 28 days

Drug: Ponatinib, 45 mg dose once per day

Sunitinib (Sutenttm; Pfizer) makes sense as a pan-growth factor receptor inhibitor; dasatinib (Spryceltm; Bristol Myers Squibb) is a Src and c-Kit inhibitor and is a reasonable choice for AML; nilotinib (Tasignatm; Novartis) is a pretty specific Bcr-Abl kinase inhibitor and is probably only being used for the ALL population – and even there only 25% of ALL patients carry this translocation; sorafenib we discussed earlier; ponatinib (Iclusigtm; Ariad) has a grab bag reactivity profile, hitting the BCL-ABL kinase, FLT3, RET, c-KIT and the FGFR, PDGFR and VEGFR growth factor receptor kinases. This is a dangerous drug, with a very narrow FDA approval in CML, and I suspect enrollment in this little exploratory trial will be stopped if possible.

If I had to guess I would say that this rather odd trial design has several goals. One is to look for signs of efficacy, although a month is pretty short duration. One might also look for patterns of resistance to therapy, which would be very interesting. Since this is BMY, I’d be surprised if they weren’t also looking at cell surface markers for possible immunotherapy treatment – more on this subject later.

Results from a dasatinib trial in CBF-AML were recently presented at the American Society of Hematology (ASH) conference (Abstract #357). Dasatinib was added to induction and consolidation chemotherapy in newly diagnosed AML patients. Unlike the rrAML population, the CBF-AML population can experience sustained periods of remission prior to relapsing, especially in younger patients. Since some of the relapses are driven by gain of function mutations in c-Kit, dasatinib should prevent at least those clones from becoming established. Early results looked good but longer term data are needed to see if this regimen will remain effective.

Imatinib (Gleevectm; Novartis) another Bcr-Abl, c-Kit and PDGF-R inhibitor, has been tested in multiple AML trials, but the results have not led to approval for use in AML. An interesting trial of the cytotoxic/immunomodulatory agent lenolidomide (Revlimidtm; Celgene) plus chemotherapy is being run by the NCI (NCT01246622). Lenolidomide has been approved for the treatment of a different bone marrow resident cancer, multiple myeloma (MM).

Anyway there is a lot of similar clinical trial work being done – using approved drugs in this new indication and looking for efficacy. This is ultimately good both for patients and the drug development companies.

Lets move on to some newer drugs in the pipeline. The FLT3 inhibitors give us a sense of the difficulty here, with low response rates as monotherapies.

Quizartinib (Ambit Biosciences; AMBI) remains stuck between phase 2 and 3 for relapsed/refractory (rr) AML. This drug is a FLT3 inhibitor with a somewhat tortured history, having been partnered for a time with Astellas, then returned, then running nicely in the clinic before running into disagreement with the FDA over approvable endpoints and safe dosage. In early December the company announced it would have to run a phase 3, likely with lower starting doses, in order to obtain FDA approval. Investors were hoping the company could file on its phase 2 trials. Notably, later in December Ambit showcased its’ quizartinib data from the FLT3-ILD rrAML trial, in which a 50% response rate (50% or greater reduction in leukemic blast cells) was reported with relatively low doses of drug. Unfortunately, it will be a while yet before more news becomes available about this drug.

In the meantime heavy hitter Novartis is already in phase 3 with its’ FLT3 and Protein Kinase C inhibitor midostaurin. The phase 3 in newly diagnosed patients is being run by the NCI (our tax dollars at work), along with The Alliance for Clinical Trials in Oncology and the Cancer and Leukemia Group (NCT00651261). A trial of midostaurin administered with or without bortezomib in adult rrAML patients is being run by Novartis and Millennium/Takeda (NCT01174888). Preliminary results were presented at ASH (abstract #3966). While response rates were impressive the toxicity was extreme, and this seemed to be due to the bortezomib dose, which was adjusted. Phase 2 trials in adult patients who carry c-KIt, FLT3-ILD, and various other mutation or cytogenetic markers are also underway (NCT01830361, NCT01846624). A phase 2b midostaurin  monotherapy study published several years ago showed modest improvement in AML patients with mutated FLT3; this study recognized the need for combination therapy to improve the clinical response (http://www.ncbi.nlm.nih.gov/pubmed/20733134).

Another FLT3 inhibitor, lestaurtinib, is the subject of 2 NCI sponsored trials in pediatric ALL/AML but drug development of this agent seems to have stalled when Teva bought Cephalon. Another FLT3 inhibitor is PLX3397 (Plexxikon) which has activity against  KIT, CSF1R and FLT3. This drug is in a phase 1/2 trial in adult rrAML (NCT01349049).

One of the major challenges for FLT3 inhibitors is breadth of action. These inhibitors work best on patients who have mutated FLT3 and are less effective in patients with normal FLT3. Also, secondary mutations have already been discovered in response to FLT3 inhibition. Specifically, in those patients who have mutations in the active site of the kinase, so-called gatekeeper mutations arise, conferring resistance to the drug.

A dominant theme in recent drug development for AML has been built on the observation that proteasome inhibitors can impact cancers of the bone marrow. Disruption of proteasome activity blocks a wide spectrum of cellular activities, and is particularly effective against rapidly dividing cells (like leukemic blasts) but also relatively quiescent tumor stem cells, that require specific proteasome-dependent signaling pathways (e.g. NK-kB). Bortezomib (Velcadetm, Millennium/Takeda) has shown activity in older patients when combined with chemotherapy. A phase 3 combination trial with sorafenib in newly diagnosed AML patients is underway, sponsored by the NCI (NCT01371981).

Carfilzomib (Onyx Pharmaceuticals) is in an early stage trial for AML, along with extensive trials in MM, B cell lymphomas, etc. The drug is furthest along in MM, now in phase 3 (NCT01568866). Early reports so far have suggested that this drug has an activity profile similar to bortezomib, but may have a better safety profile. This is an interesting drug (and company) to watch. They have a second generation oral version of carfilizomib, oprozomib, in phase 1 MM trials. Millennium/Takeda are developing ixazomib in MM and lymphomas. An AML trial is listed but not yet recruiting.

A third theme that we can follow in AML therapeutic drug development is the use of drugs that impact epigenetic gene regulation. Because AML is driven by genetic translocations, gene regulation at the level of chromatin structure is disrupted. There are two processes at work here that can be targeted. One is the aberrant methylation of CpG islands in gene promoter regions, which can be targeted by DNA methyltransferase inhibitors. The second is changes in the conformation of chromatin caused by dysregulated histone acetylation. This process can be therapeutically targeted using histone deacetylase [HDAC] inhibitors.

The HDAC inhibitor vorinostat (Zolinzatm, Merck) has been extensively studied in AML, and is currently in a phase 3 trial with chemotherapy for young patients with newly diagnosed disease (NCI; NCT01802333). Vorinostat monotherapy was generally ineffective, but combination with chemo agents proved much more potent. As detailed at ASH in December (Abstract #2684), newly diagnosed and rrAML patients were enrolled in a phase 2 expansion study. Of 75 patients, 57 patients achieved CR, and 7 achieved CR with incomplete platelet recovery (CRp), for an overall response rate of 85 percent. Median overall survival was 82 weeks and median event free survival was 47 weeks. For patients with the high-risk Flt-3 ITD mutation the 10/11 achieved CR and 1/11 CRp. The ORR = 100% in these patients. Their median overall survival was 91 weeks and median event free survival was 66 weeks. About 25% of the total patients in CR received SCT.

Other combination trials include the sorafenib trial mentioned above, and a trial in combination with antibody therapy (gemtuzumab ozogamicin) for rrAML (NCI; NCT00895934). This trial reported early results at ASH (Abstract #3936). The response rates ere encouraging and about 20% of patients obtained durable remission. There were significant toxicity issues. This drug is very likely to play a critical role in the evolution of combination therapy for AML. We’ll discuss antibody therapies further in Part 2.

Other important HDAC inhibitors in development for AML is panobinostat (Novartis). What’s interesting about the development campaign with this drug is the pairing in multiple trials with 5-azacitidine, a DNA methytransferase inhibitor. In such settings two modes of epigenetic regulation are being targeted simultaneously. One of these studies published findings last month                                 (http://www.nature.com/bcj/journal/v4/n1/full/bcj201368a.html) and demonstrated good tolerability and reasonable response rates. Clearly, this combination should move forward in the context of chemotherapy or other drugs. Of note the DNA methyltransferase inhibitor decitabine (Dacogentm, MGI Pharma) is already approved for AML. There was also a presentation on the HDAC inhibitor entinostat (Syndax Inc) with 5-azacitidine in myeloid neoplasia (Abstract #2777), and there are several clinical trials listed for AML, however this drug is mainly being used in solid tumor trials.

Other interesting drugs in this area include alisertib, an Aurora A kinase inhibitor (Millennium/Takeda) being tested extensively in B and T cell lymphomas and in solid tumors. There are several AML trials including a phase 2 trial completed by MLMN (NCT00830518). Selinexor, (Karyopharm) a selective inhibitor of nuclear export, in in phase 1 trial for advanced AML. Abbvie’s Bcl2 inhibitor ABT-199 is also in an AML trial.

If we take a step back we can appreciate that in small molecule development Novartis, Merck and Onyx are placing big bets in this therapeutic area. We’ll sort out the best looking therapeutics as we dig in a little deeper.

In Part 2 we’ll take a look at the biologics landscape, and begin to draw the bigger picture.

Conference Update: Targeting the Wnt Pathway in Oncology

drug mechanism of action, Pathological Cells and Processes, Pathways for Drug Development, Wnt

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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.

A Science-Side Guide to the New Oral Multiple Sclerosis Drugs

Aubagio, drug mechanism of action, Gilenya, MS, multiple sclerosis, Tecfidera

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Back to the blog now that I’ve settled into a new career (more on this another day).

Last week brought a wave of commentary on the evolving Multiple Sclerosis (MS) marketplace, as the European Medicines Agency (EMA) rendered positive opinions for two new oral drugs for the treatment of relapsing and remitting MS: Tecfidera (dimethyl fumarate or BG-12) and Aubagio (teriflunomide). FDA approval of Tecfidera is expected this week; Aubagio won FDA approval in September 2012. The focus of much of the discussion was the impact these new drugs would have on the companies that dominate the MS market: Biogen Idec, Novartis, Sanofi/Genzyme, Teva, EMD-Serono, and Takeda/Millenium. A summary of last week’s approvals can be found at FierceBiotech: http://tinyurl.com/cekba9r.

The consensus is that these new drugs will very quickly impact MS treatment paradigms and alter the fortunes of companies operating in this market. This is therefore a particularly good time to have a look behind the hype, and review the scientific rationale behind some of these new therapies. I’ll address the landscape with one eye on drug efficacy and one on known and potential side-effects of this new class of MS therapeutics.

The current stage was set in September 2010 with the approval of Gilenya (fingolimod), an S1P receptor modulator developed by Novartis. S1P receptors regulate a bewildering array of biological and pathological responses. Gilenya acts on at least 4 different S1P receptors, so formally speaking its’ mechanism of action in MS is undefined. It does appear however that the basis for the efficacy of this drugs lies in its ability to down-regulate the activity of the S1P receptor 1 (S1P1). S1P1 has many functions, including regulating the exit of lymphocytes, particularly T cells, from lymph nodes. Since all T cells that are in circulation will move through both the bloodstream and the lymphatic system, they all move through lymph nodes, which are organs that lie within lymphatic circulation. As T cells become trapped in lymph nodes, the number of circulating T cells in the bloodstream drops precipitously. In MS, autoreactive T cells use the blood circulatory system to gain access to the central nervous system (CNS) and attack myelin and other CNS antigens, thereby causing the disease. Gilenya is effective in MS because it prevents T cells from reaching the CNS, instead trapping them inside lymph nodes. This mechanism of action (MOA) is superficially similar to that of Biogen Idec’s Tysabri (natalizumab), a biologic drug that acts on T cells by preventing their exit from the bloodstream into the CNS (and other tissues). Both drugs therefore impact MS via effects on T cell movement. Tysabri is the single most efficacious drug developed for MS capable of reducing the annualized relapse rate by 68%. However, use of this biologic drug requires intravenous infusion (IV) and extended therapy is associated with a variety of side-effects, including the very dangerous disease PML. Its fair to say that Biogen-Idec has worked very hard at defining and managing PML risk associated with Tysabri use, and Tysabri continues to be a dominant drug in this market. I’ll come back to the efficacy and side-effect profiles of Gilenya in a bit, but first lets introduce the newer drugs.

Tecfidera was developed by Biogen Idec and is their first oral drug for MS. On March 20th the EMA’s Committee for Medicinal Products for Human Use (CHMP) issued a positive review of Tecfidera as a first-line therapeutic for the treatment of relapsing remitting MS. A positive CHMP opinion means that the drug is likely to be approved by the EMA for sale in the EU within a few months. FDA approval for use in the US is expected on March 28th. Tecfidera, like Gilenya, does not have a formally defined MOA, although there is good evidence to support the hypothesis that this drug primarily acts as an NRF2 activator. This is a compelling MOA, as NRF2 is a master regulator of the oxidative stress response. The drug may very well act on both the inflammatory response to myelin and other CNS proteins, blunting its intensity and impact, and directly on cells within the CNS, via cytoprotective effects. The efficacy of this drug was demonstrated in 2 large phase III studies (DEFINE and CONFIRM) and is impressive. Pooled phase III data showed reduced disease burden as measured by annual relapse rate (reduced by 49%) and identification of new or enlarged MS lesions (reduced by 78%) among other favorable outcomes. Equally compelling is the safety profile of this drug, with side-effects generally limited by unpleasant gastrointestinal (GI) symptoms including nausea, diarrhea and abdominal pain. For many patients these side effects wane after the first month.

Aubagio was developed by Sanofi and its’ subsidiary Genzyme. Aubagio is the teriflunomide metabolite of leflunomide, an approved drug for rheumatoid arthritis. These drugs are pyrimidine synthesis inhibitors that function by targeting the mitochondrial enzyme dihydro-orotate dehydrogenase. Inhibition of pyrimidine synthesis blocks DNA replication and causes cell death of dividing cells like activated T cells and B cells, thereby reducing lymphocyte proliferation. Not surprisingly, this class of cytotoxic drugs was first developed in the context of cancer therapy.

So with Aubagio we are back to effects on lymphocytes, presumably impacting activated T cell survival and function, reminiscent of Tysabri and Gilenya that also target T cell activity. Aubagio has a rather different efficacy/toxicity profile than the other drugs we have discussed so far. With a risk reduction of approximately 30% in annualized relapse rate, Aubagio has an efficacy profile similar to the beta interferons (Avonex, Betaseron, Rebif) and Copaxone, drugs that are delivered by cutaneous injection. On this basis, Aubagio would benefit from the fact that it is an oral medication, not requiring injection. The safety profile for Aubagio is perhaps more problematic, as this drug carries a black box warning for liver toxicity and is contraindicated in women who are pregnant or are likely to become pregnant due to concerns about teratogenicity. Also Aubagio is associated with increased susceptibility to infections, alopecia (hair loss) and GI effects.

Despite the safety concerns, Aubagio has attracted use among US neurologists whose patients want an oral medication, and this is despite the modest efficacy profile. This brings us back to Gilenya, the first oral MS drug approved in the US and EU. Gilenya is certainly efficacious, with a reduction in the annualized relapse rate of 54%, which is very similar to Tecfidera. Gilenya has a challenging risk profile, with a still poorly understood cardiovascular risk and a high rate of opportunistic infections. Recently, 15 unexplained patient deaths have triggered a review by the EMA that could lead to new safety warnings. Bradycardia continues to be an issue with Gilenya use, requiring patient monitoring after the first dose for at least 6 hours. Despite this safety profile, the efficacy of Gilenya has driven substantial use, and Novartis reported at the American Association of Neurologists meeting last week that no new toxicities have been seen in long-term extension studies.

It’s worth briefly mentioning two other potential oral medications for MS. Laquinimod is an immunomodulator from Teva and Active Biotech. Laquinimod’s MOA is not well understood. The drug has given mixed results in 2 phase III MS clinical trials, with pooled data showing only a 21% reduction in annualized relapse rate. A third trial is underway. The drug appears to be relatively safe, and may be appropriate for some patients although the limited efficacy will make wide use of this drug difficult to justify. Cladribine, from Merck Serono, is another drug that interferes with DNA metabolism and is broadly cytotoxic. It was withdrawn from consideration for use in MS after FDA rejection and negative EMA guidance. Cladribine was associated with an array of severe toxicities consistent with its cytotoxic MOA, including high risk of infection, neutropenia, liver toxicity, effects on the CNS and other side effects.

What we see then among the class of oral MS drugs is a spectrum of efficacy and toxicity profiles that will determine the evolution of their use in the context of existing injectable drugs. We see distinct mechanisms of action that will allow for class differentiation and, perhaps, for combination therapy. This latter goal, likely to be a critical development to stopping MS progression completely, will be achieved if, and only if, the toxicity profiles of potential combination therapies allow. In this regard the use of injectable beta interferons may be most compatible with the use novel orals like Tecfidera. A few combination therapy trials are underway.

Watch this space for further updates on developments in autoimmunity and oncology, my favorite subjects in drug development.

Please follow the author on Twitter @PDRennert and connect on LinkedIn.

Sugar cones: perfect content and analysis

Companies - stock symbols, drug mechanism of action, Drugs (generic and brand names), Pathological Cells and Processes, Pathways for Drug Development

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Introducing the Sugar Cone Biotech Blog

When we consider sources of biotech and pharma intel and insight, we see striking differences in style. Lets look at two very common types of content, then offer an alternative.

Style 1: Wafer Cone Analysis. Short on insight, full of reposts and recycled story lines. This is the most common form of biopharma “news” and clogs the web with redundant traffic. Apt to show up uninvited in your inbox, the Wafer Cone wraps just enough fluff around a bit of information to give the appearance of substance while adding nothing of its own. Cheap or free, of little value regardless.

Style 2: Waffle Cone Analysis. Always too much of a good thing, layering heavy “added value” to otherwise straightforward analysis. Bloated and expensive, the Waffle Cone is the speciality of large consulting houses that churn out commercial analyses of technology development, of clinical stage therapeutics, of medical practice and of the competitive landscape. Characteristics include broad consensus with client’s existing assumptions, a ‘tell ’em what they want to hear’ approach to analysis, and the selective use of supportive quotes from KOL’s. Add a sprinkle of the latest business buzz words and you’ve got it. And its going to cost you plenty.

These are obviously extremes on the spectrum that spans biopharma news, blogs and consulting.

I’d like to define a better approach:

Sugar Cone Analysis. A problem-driven approach designed to capture essential data and provide critical insight to enable action. No more, no less. We track critical trends in drug development, medical research and practice, translational efforts, clinical trial design, and breakthrough technologies. All posted here for open commentary. There are already many excellent science and business blogs that capture content (see who we follow), but we push analysis to generate hypotheses: what will or should happen next. And we’ll check back in to see how our hypotheses fare in the real world.

Through Clarion Bio Consultancy we offer our clients transparent, critical data analysis and its’ impact on the questions at hand. We specialize in helping biotech and academic groups build out research programs and investment clients build out companies. We focus on creating research plans the drive asset momentum, staging programs for investment, and critical assessment of existing programs. We ensure upfront that the questions, underlying assumptions and timelines are clear. Iterative communication ensures alignment on goals and progress. Outcomes and recommendations are presented without prejudice, allowing confident and crisp action.

Contact us for more information: rennertp@sugarconebiotech.com

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