The 30 September issue of Nature included a major emphasis on translational research in cancer. Featured articles included an editorial, a Perspective, and a research report. There was also an online “Specials” archive on translational cancer research, containing many recent research reports, all with free access to nonsubscribers.

The theme of these articles was the development of novel strategies for accelerating the translation of research on cancer biology into safe and efficacious therapies.

The Perspective, entitled “Translating cancer research into targeted therapeutics”, by British researchers Johann S. de Bono, M.D., Ph.D. and Alan Ashworth, Ph.D., outlines a novel disruptive clinical trial strategy for accelerating the translation of biology-driven oncology drug discovery into the clinic, with an early determination of proof of concept. This new strategy is designed  to ameliorate the high levels of Phase 2 and Phase 3 attrition of cancer drugs, as well as to lower the cost of clinical trials and to shorten the time from preclinical studies to the approval and marketing of oncology drugs that successfully emerge from clinical trials. It also is designed to aid in the development of therapies that provide greater patient benefit (in terms of progression-free survival) than the typical new oncology drugs that reach the market.

We have discussed clinical trial strategies of this type in two of our publications–our 2009 book-length report Approaches to Reducing Phase II Attrition (available from Cambridge Healthtech Institute, and our 2009 article published in Genetic Engineering and Biotechnology News, “Overcoming Phase II Attrition Problem” (available on our website). The de Bono and Ashworth article provides a more detailed and specific presentation of this strategy with respect to oncology drug development, and provides several examples of its successful application.

In the traditional Phase 1/Phase 2/Phase 3 format of cancer drug development, clinical studies focus on treating patient populations with advanced cancers that have not been characterized in terms of their genetics and molecular biology. The trials culminate in large, pivotal randomized Phase 3 trials that typically last several years, and are aimed at regulatory approval. In most cases, new drugs that emerge from Phase 3 trials and win approval only improve survival by a few months. Patients who participate in Phase 1 and Phase 2 clinical trials usually derive little or no benefit, and a high proportion of drugs fail in Phase 2 or Phase 3. This clinical trial format determines how well a particular drug or drug combination works for the average patient. However, that treatment might not be the best for a given individual patient.

As basic researchers have advanced the study of molecular genetic pathways of cancer, drug discovery researchers have been developing targeted therapies for cancer. These drugs–such as monoclonal antibodies (MAbs) like trastuzumab (Genentech/Roche’s Herceptin) and kinase inhibitors like imatinib (Novartis’ Gleevec/Glivec) work by modulating specific biomolecules (e.g., overexpressed or mutated oncogenic proteins) that are critical for the malignant phenotype. Population-based clinical trials of unselected patients make little or no sense in developing targeted therapies. Instead, clinical researchers need to first select groups of patients whose cancers express the biomolecule to be targeted, and preferably whose cancers are driven by that particular biomolecule. This way of thinking leads to the formulation of a new clinical trial strategy, as outlined in the Perspective.

Drs. de Bono and Ashworth call this strategy a personalized medicine (or “stratified medicine”) hypothesis-testing approach. The first step in this strategy is to develop a strong biological hypothesis that a particular altered molecular target is critical for the malignant phenotype of a particular cancer. This hypothesis is usually generated as the result of laboratory and clinical studies. Researchers need to show that blocking of the function of the altered target results in a lethal or cytostatic effect in cancer cells that express the the target, but not in normal cells that do not. It is also preferred that resistance to agents that block the target is not easily gained.

In the discovery stage of this strategy, researchers need not only to identify and optimize clinical candidate drugs that modulate the target, but also biomarkers that can be used to identify patients whose tumors express the altered target and are therefore likely to benefit from treatment. These biomarkers are called “enrichment biomarkers”, and have the potential to become predictive biomarkers. (Predictive biomarkers may also be the basis for the development of companion diagnostics). It is also important to identify pharmacodynamic biomarkers (biomarkers that can be used to determine target occupancy by the drug) and intermediate endpoint biomarkers (which can assess antitumor activity of the drug–for example, radiological assessment of tumor regression). Identification and qualification/validation of biomarkers is a work in progress, and is a critical need for further development and utilization of the personalized medicine hypothesis-testing clinical trial strategy.

Once targets and drugs that modulate them have been identified, they must be validated in animal models. The issue of the inadequacy of current mouse models of cancer–mainly xenograft models in which human cancer cell lines are transplanted into immune deficient mice–to predict drug efficacy is important both in the traditional cancer drug development strategy and in the novel strategy discussed in the de Bono and Ashworth article. We have discussed development of improved animal models for cancer drug development in an earlier blog post. de Bono and Ashworth note that there is an urgent need to develop such improved animal models. Nevertheless, as discussed in the de Bono and Ashworth article, there are examples of the successful implementation of the personalized medicine hypothesis-testing strategy of cancer drug development that have used traditional animal models in the preclinical phase.

In the personalized medicine hypothesis-testing strategy, clinical trials have the same three phases as in traditional trials. However, the trials involved stratification of patients using biomarkers, such that clinical studies are done in patients whose tumors express the target of the drug. Trial design is also more flexible and adaptive, and is contingent on obtaining key clinical data. The trials focus on determining the following as early as possible:

  • Proof of mechanism: determining a dose range and dosing schedule under which the drug achieves sufficient target occupancy for long enough, using biomarker-based pharmacodynamic assays.
  • Proof of concept: Determining that once sufficient target occupant is achieved, the drug exhibits antitumor activity, as determined using intermediate endpoint biomarkers.

In first-in-human clinical trials, in addition to determining safety and tolerability and evaluating pharmacokinetics and pharmacodynamics as in traditional Phase 1 trials, researchers also pursue rapid dose escalation, until proof of mechanism is achieved, using the appropriate biomarkers. Researchers then move on to proof of concept hypothesis testing, at doses and dosing schedules (ideally, the maximum tolerated dose) that are sufficient to address the target for long enough to have a biological effect. Ideally, researchers should move seamlessly from determination of proof-of-mechanism to assessment of antitumor activity, via adaptive trial design and patient selection using enrichment biomarkers.

If the above early-stage strategy results in a strong determination of proof of concept, this provides the basis for moving on to Phase 3 trials in patients selected using enrichment/predictive biomarkers, with the goal of drug registration. Such Phase 3 trials should have a higher probability of success than traditional Phase 3 trials in unselected patient populations, with less than adequate demonstration of proof of concept in Phase 2.

However, in the personalized medicine hypothesis-testing strategy, there is also the need for reiterative translational studies, between the laboratory and the clinic and back to the laboratory. Such studies should be designed as early as possible in clinical development. For example, clinical trials might allow researchers to obtain tumor samples to determine mechanisms of drug resistance. Such studies might form the basis for generating further hypotheses that are relevant to reversing drug resistance, via such means as development of combination therapies or of second-generation drugs.

The personalized medicine hypothesis-testing strategy is a work in progress. However, as we shall discuss in Part 2 of this series, there are examples of its successful implementation. And this strategy provides a means to extend biology-driven drug discovery, arguably the most successful drug discovery strategy of the past decade, into early and mid-stage clinical trials, thus increasing the probability of clinical success.

Nevertheless, it must also be emphasized that our understanding of disease biology (especially cancer biology) is limited, thus limiting our ability to successfully carry out biology-driven drug discovery in all cases. However, as our understanding of disease biology grows–in an incremental manner–as the result of basic research mainly in academic laboratories, we should be able to utilize this research to develop novel, breakthrough treatments via biology-driven drug discovery and personalized medicine hypothesis-testing clinical trials.


In March 2010, we published two articles on this blog relating to Roche/Plexxikon’s PLX4032 for metastatic melanoma. The first article, dated March 2, described a Phase I clinical trial of the drug, based on an article about this trial in the New York Times (NYT). The second article, dated March 10, described Plexxikon’s discovery of PLX4032, using its proprietary “scaffold-based drug design” technology platform. The latter post is among the most popular articles on this blog.

Now the results of the Phase I trial of PLX4032 has been published in the August 26, 2010 issue of the New England Journal of Medicine (NEJM). (A subscription is required to read the full article.)

As we discussed in our previous articles, PLX4032 is a B-Raf (called “BRAF” in the NEJM paper and in some other publications) kinase inhibitor that is exquisitely specific for B-Raf carrying the V600E mutation. B-Raf(V600E) is the most common somatic mutation found in human melanomas. Researchers believe that B-Raf(V600E) is a “driver mutation” that is particularly critical for the malignant phenotype of human metastatic melanomas that carry the mutation. B-Raf(V600E) is constitutively activated, and melanomas carrying this mutation can proliferate independently of growth factor signaling, resulting in the runaway proliferation characteristic of the malignant phenotype.

The clinical trial described in the NEJM article was carried out by researchers at Plexxikon and Roche, in collaboration with academic researchers at five institutions in the United States and Australia. The trial was led by Keith T. Flaherty, M.D. (then at the University of Pennsylvania in Philadelphia, and now at the Massachusetts General Hospital Cancer Center [where he is Director of Developmental Therapeutics] and the Dana-Farber Cancer Institute in Boston) and Paul B. Chapman, MD (Memorial Sloan-Kettering Cancer Center).

As discussed in the NEJM article, the researchers conducted a multicenter Phase I dose-escalation trial of PLX4032 (which is orally available), followed by an extension phase in which patients were given the maximum dose that could be administered without adverse effects (960 mg twice daily). (The latter dose is the recommended Phase II dose.) A total of 55 patients (49 of whom had melanoma) were enrolled in the initial, dose-escalation portion of the trial. 32 additional patients, all of whom had metastatic melanoma with the B-Raf(V600E) mutation, were enrolled in the extension phase. Patients were given the drug twice a day until they had disease progression.

In the dose-escalation phase, among the 16 patients with melanoma carrying the B-Raf(V600E) mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response (i.e., tumor shrinkage of at least 30%) and 1 had a complete response. Among the 32 patients in the extension cohort, 24 had a partial response and 2 had a complete response. The latter figure represents an 81% response rate. The estimated median progression-free survival among all patients was over 7 months.

Dose-limiting adverse effects included rash, fatigue, and joint pain.

The published results of this Phase I trial elicited great enthusiasm in the popular press and in such industry media as Fierce Biotech and BioWorld Online, and by oncologists who were interviewed for these articles. The oncologists said that they had never seen such a dramatic response in treatment of metastatic melanoma.

Because PLX4032 is targeted to a specific oncogenic mutation, Plexxikon and several industry commentators refer to the use of the drug as an example of personalized medicine. In parallel with development of PLX4032, Plexxikon and Roche Molecular Systems are developing a DNA-based companion diagnostic to identify patients whose tumors carry the B-Raf(V600E) mutation.

PLX4032 is on an accelerated path to potential registration. Parallel Phase II and Phase III clinical trials are in progress in previously treated and previously untreated patients, respectively, all who have metastatic melanoma carrying the B-Raf(V600E) mutation.

Meanwhile, the results of a Phase III trial (in 676 patients with advanced melanoma) of Medarex/Bristol-Myers Squibb’s (BMS’s) ipilimumab were published in the August 19, 2010 issue of the NEJM.  Ipilimumab, unlike the targeted therapeutic PLX4032, is an immunomodulator that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) to potentate an antitumor T-cell response. Ipilimumab is a monoclonal antibody, unlike PLX4032 which is a small-molecule compound. In this NEJM article, the researchers reported that ipilimumab–given with or without the gp100 peptide vaccine–showed a median overall survival of 10 months, as compared to 6.4 months in patients receiving gp100 alone. Ipilimumab treatment also gave improved one-year survival compared with gp100 alone–46% versus 25%. Two-year survival was 24% in the ipilimumab group and 14 percent in the gp100 group. BMS has filed a Biologics License Application (BLA) for ipilimumab, and earlier this month (August 2010) received fast-track status from the FDA for the drug.

Ipilimumab treatment is associated with autoimmune toxicities (especially enterocolitis), which can be severe. These are usually reversible by treatment with high-dose steroids.

Decision Resources published our report on development of immunomodulators in treatment of cancer in 2007. This report includes a discussion of ipilimumab, and provides further information on its mechanism of action, adverse effects, etc., as well as on other immunomodualtors for treatment of cancer, some of which are now on the market.

We believe that it is important to pursue development of both targeted therapies and of immunomodulators for metastatic melanoma. This may provide oncologists a range of therapeutics (and of combinations of therapeutics) to treat this disease, which now has very few treatment options and a very poor prognosis.

The results with both PLX4032 and ipilimumab provide hope for better treatment of at least some classes of metastatic melanoma in the near future. However, as discussed in our March 2010 articles, even in the case of PLX4032 treatment of melanoma carrying the B-Raf(V600E) mutation, it will most likely be necessary to develop combination therapies in order to achieve long-lasting remissions or cures.

In the December 15, 2009 issue of Neurology, a research report by Stephen Salloway and his colleagues at the Butler Hospital and Brown University (Providence, RI) and an editorial by Dan Kaufer and Sam Gandy (University of North Carolina at Chapel Hill) focus on a Phase II multicenter placebo-controlled clinical trial of Elan/Wyeth’s bapineuzumab (AAB-001) in patients with mild to moderate Alzheimer’s disease (AD). (Wyeth is now part of Pfizer.) (A subscription is required to read the full text of both of these articles.) Bapineuzumab is a monoclonal antibody (MAb) drug that is specific for amyloid-β (Aβ) peptide. The dominant paradigm among AD researchers and drug developers is that the disease is caused by aberrant metabolism of Aβ, resulting in accumulation of neurotoxic Aβ plaques. This paradigm is known as the “amyloid hypothesis”.

The overall result of the study by Salloway et al. was that there was no difference in cognitive function between patients in the drug-treated and the placebo groups. However, the study did not have sufficient statistical power to exclude the possibility that there was such a difference. About 10% of patients treated with the agent also experienced vasogenic edema (VE), which was reversible. (Cerebral VE is the infiltration of intravascular fluid and proteins into brain tissue, as the result of breakdown of the blood-brain barrier.)

Retrospective analysis of the data suggested that bapineuzumab-treated patients who were not carriers of the apolipoprotein E epsilon4 allele (ApoE4) showed improved cognitive function as compared to placebo treatment, and that they had a lower incidence of VE than ApoE4 carriers. The ApoE4 polymorphism is the only known, well-characterized genetic risk factor associated with the development of late-onset AD. Of the three common isoforms of ApoE, ApoE3 is the most common, followed by ApoE4 and ApoE2, respectively. Unlike ApoE4, the ApoE2 allele appears to protect against development of AD. Some researchers estimate that allelic variations in ApoE may account for over 95% of AD cases.

In the study by Salloway et al., nearly two-thirds of the AD patients carried one or more ApoE4 alleles; thus only the remaining one-third of patients appeared to show positive effects of bapineuzumab treatment according to the retrospective analysis. However, the idea that the drug is efficacious in ApoE4 noncarriers is only a hypothesis, which will require prospective clinical trials to confirm. Elan and Pfizer are now conducting large Phase III clinical trials of bapineuzumab, which have prospectively segregated enrollment into ApoE4 carrier and noncarrier groups.

The hypothesized association of ApoE4 noncarrier status of AD patients with bapineuzumab efficacy and safety has been used as a case study in workshops on stratified medicine sponsored by the FDA, MIT, and industry partners in 2009 and 2010. You can read about the October 2009 workshop here. The most recent workshop was held at MIT on January 19, 2010. In these workshops, two case studies were discussed: the use of diagnostic tests for the HER2 receptor in identifying breast cancer patients who are likely to benefit from treatment with trastuzumab (Genentech/Roche’s Herceptin), and the bapineuzumab/ApoE4 case. The HER2/ trastuzumab relationship is well known and well characterized, and is considered to be a paradigm of stratified medicine. This contrasts with the bapineuzumab/ApoE4 association, which remains a hypothesis pending the results of the Phase III prospective clinical studies.

A growing minority of researchers is skeptical that the amyloid hypothesis is sufficient to account for AD pathogenesis in all stages of the disease or in various disease subpopulations, and they are investigating other pathways that may contribute to the disease, either in combination with the amyloid pathway or as alternative mechanisms. We have discussed alternative hypotheses for AD pathogenesis in a 2004 article published in Genetic Engineering News (available on our website), and in book-length reports published by Cambridge Healthtech Institute in 2006 and in 2009.

The search for alternative hypotheses takes on added urgency because of the clinical failure of several AD drugs that had been designed based on the amyloid hypothesis. These include Neurochem’s (now Bellus Health) Alzhemed (3-amino-1-propanesulfonic acid) and Myriad Pharmaceuticals’ Flurizan (tarenflurbil), both of which failed in Phase III clinical trials. Based on the overall results of the Phase II trial of bapineuzumab, most researchers and industry commentators would add bapineuzumab to the list, unless the stratified Phase III trial shows that the drug is significantly efficacious and safe for ApoE4 noncarriers.

Since ApoE4 carrier status is such a prominent risk factor for developing late-onset AD, might ApoE4 itself be a target for drug discovery in AD? Drs. Kaufer and Gandy suggest that such an approach might be fruitful, whatever the outcome of the Phase III trial of bapineuzumab. Several academic laboratories have been investigating mechanisms by which ApoE4 may be involved in the pathobiology of AD. You may read two recent papers on this subject here and here. ApoE4 may contribute to AD pathogenesis via multiple mechanisms, including by causing synaptic deficits and mitochondrial dysfunction in neurons, and by inducing endoplasmic reticulum stress leading to astrocyte dysfunction.

Given the prominence of ApoE4 expression as a risk factor for AD, the study of the mechanistic basis of ApoE4’s role in AD pathobiology needs greater attention. Hopefully, this research will lead to the development of novel therapeutic strategies for AD.

In the December 10 2009 issue of Nature, researchers at Agios Pharmaceuticals (Cambridge, MA) and their academic collaborators published an article implicating mutations in a metabolic enzyme, cytosolic isocitrate dehydrogenase (IDH1) as a causative factor in a major subset of human brain cancers.

The mutated forms of IDH1 are found in around 80% of human grade II-III gliomas and secondary glioblastomas. The mutations occur in arginine 132, which is usually mutated to histidine. (In other less common mutations, arginine 132 is mutated to serine, cysteine, glycine, or leucine.) Typically, only one allele of IDH1 is mutated. These mutations appear to occur early in the process of tumorigenesis, and often appear to be the first mutation that occurs. The mutant forms of IDH1 are also found in a subset of acute myelogenous leukemia (AML).

The wild-type form of IDH1 catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate to α-ketoglutarate. However, the researchers found that the mutant forms of IDH1 no longer catalyzes this reaction, but instead catalyzes the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). This is the result of changes in the active site of the enzyme, as demonstrated by structural studies carried out by the researchers. Tumors that harbor the mutant form of IDH1 have elevated levels of 2HG. The researchers therefore hypothesize that these elevated levels of 2HG are a causative factor in tumorigenesis and/or tumor progression in human gliomas.

This hypothesis is supported by the effects of the familial metabolic disorder 2-hydroxyglutaric aciduria. This disease is caused by a deficiency of 2-hydroxyglutarate dehydrogenase, an enzyme that converts 2HG to α-ketoglutarate. Patients with this metabolic disease have elevated levels of 2HG in bodily fluids and in the brain, and an increased risk of developing brain tumors.

The mechanism by which 2HG might contribute to tumorigenesis is unknown. The authors advance several hypotheses, including increasing reactive oxygen species (ROS) levels, serving as an NMDA (N- methyl-D-aspartate) receptor agonist, and competitive inhibition of enzymes that use glutamate and/or α-ketoglutarate resulting in the induction of hypoxia-inducible factor-1α, a transcription factor that facilitates tumor growth under conditions of hypoxia.

According to the authors, these results suggest that in patients with low-grade gliomas containing mutant forms of IDH1, therapeutic inhibition of 2HG production may slow or halt progression of these tumors to lethal secondary glioblastomas. 2HG levels may also be used as a prognostic test for IDH1 mutations, since patients with these mutations tend to live longer than patients with gliomas that have other mutations.

The company that led this research, Agios Pharmaceuticals, is developing a pipeline of oncology drugs based on targeting metabolic pathways in cancer cells. Interestingly, Agios means “holy” in Greek.

Way back in 1924, Otto Warburg demonstrated a difference between cancer cells and normal adult cells in glucose metabolism. In the presence of oxygen, most normal adult cells metabolize glucose to pyruvate via the process of glycolysis, generating two molecules of ATP (the energy currency of the cell) per glucose molecule. In the mitochondria, they then utilize oxygen to catabolize pyruvate to CO2 and water, in the process generating 36 molecules of ATP per glucose molecule. Cancer cells, however, predominantly carry out aerobic glycolysis, in which they carry out glycolytic conversion of glucose to pyruvate, followed by reduction of pyruvate to lactate. Despite the presence of oxygen, cancer cells generate the bulk of their ATP from glycolysis, not mitochondrial oxidative phosphorylation, in the process consuming large amounts of glucose. The reliance of cancer cells on aerobic glycolysis for their metabolism is known as the “Warburg effect”.

Agios’ platform is based in part on the work of signal-transduction pioneer Lewis Cantley (Beth Israel Deaconess Cancer center/Harvard Medical School, Boston MA). It is Dr. Cantley’s work on the connection between growth factor-mediated signal transduction and aerobic glycolysis that is the basis for Agios’ platform. In particular, Dr. Cantley and his colleagues found that pyruvate kinase M2 (PKM2) is a link between signal transduction and aerobic glycolysis. PKM2 binds to tyrosine-phosphorylated signaling proteins, which results in the diversion of glycolytic metabolites from energy production via mitochondria oxidative phosphorylation to anabolic processes required for rapid proliferation of cancer cells.

Agios closed a $33 million Series A financing in July 2008, co-led by Third Rock Ventures, Flagship Ventures and ARCH Venture Partners. In June 2009, Fierce Biotech named Agios to the 2009 FierceBiotech “Fierce 15” list. On December 21, 2009, Agios received funding from the nonprofit organization Accelerate Brain Cancer Cure (ABC2), to supplement Agios’s research on the development of IDH1-based therapeutics and diagnostics. Agios expects to have a lead compound in the clinic some time in 2010.

The Agios website calls cancer metabolism “one of the most exciting new areas of cancer research”. But the study of cancer metabolism, and especially the Warburg effect, is not new—the Warburg effect is a classic observation going back 85 years. Moreover, biotechnologists working in such areas as production of recombinant proteins in CHO cells have been familiar with aerobic glycolysis, which is carried out by most mammalian cell lines in culture, for decades. Nevertheless, cancer metabolism has been well out of the mainstream of cancer drug discovery. It was Dr. Cantley’s work, which links the classic Warburg effect to the mainstream area of signal transduction and protein kinases, which has made Agios’ platform possible.

Similarly, it was Julian Adams’ work on the biology of the proteasome in the 1990s, through a series of biotechnology company mergers that eventually led him to Millennium Pharmaceuticals (now Millennium: The Takeda Oncology Company), which resulted in Millennium’s proteasome inhibitor Velcade (bortezomib). Velcade, the only proteasome inhibitor on the market, is now approved by the FDA for the treatment of multiple myeloma and mantle cell lymphoma. Prior to Dr. Adams’ work, proteasome biology and protein degradation were out of the mainstream of cancer drug discovery. Now Joseph Bolen, the chief scientific officer of Millennium, sees “protein homeostasis” as one of the most exciting areas of cancer research.

Finally, although the development of protein kinase inhibitors to target signaling pathways in cancer is now well within the mainstream of oncology drug discovery, prior to the discovery and development of imatinib (Novartis’ Gleevec/Glivec) (approved by the FDA in 2001), specific targeting of protein kinases was though to be unlikely, since all of these enzymes have a high degree of similarly in their ATP binding sites. Thus the field of protein kinase inhibitors did not enter the mainstream until the late 1990s-early 2000s.

The take-home lesson is that drug developers may find fertile areas for innovation in seemingly obscure or out-of-the mainstream areas of biology (or of chemistry, as we have discussed in previous blog posts). Some of these areas may be technologically premature, and not quite ready for exploitation by drug developers. However, as demonstrated by our blog post on monoclonal antibodies, even some technologically premature areas may yield to innovators who are willing and able to develop enabling technologies to move these areas up the development curve.