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Feature Articles : Dec 1, 2008 ( )
Zeroing in on PI3K Pathway
Myriad of Groups Are Working on the Latest Hot Target and Most Are Optimistic About Its Potential
Although the term “signal transduction” was coined back in 1972, cell-signaling switches have only recently become one of the hottest targets in cancer research. The latest star is the phosphoinositide 3-kinase (PI3-kinase) pathway—playing key roles in angiogenesis, cell growth, differentiation, and survival, as well as glucose metabolism. It is part of the PI3/AKT/mTOR pathway, which also plays a key role in cancer. Studies have shown that inhibiting this pathway suppresses tumor growth. Presenters at AACR’s conference on “Targeting the PI3-Kinase Pathway in Cancer” held last month in Cambridge, MA, provided insights into their ongoing research.
Armed with the knowledge of Lilly’s PI3K inhibitor, LY294002, researchers at Semafore Pharmaceuticals realized they could utilize their medicinal-chemistry expertise to make their own inhibitor.
Making a Better Molecule
“If we could create a pro-drug that would overcome its deficiencies, use its inherent activity, and deliver it to the tumor in a selective manner, we had the potential to make it a drug,” explained Joseph Garlich, Ph.D., CSO. PI3 kinase “has been found to be sort of the master control switch in signaling, and it is an exciting target in oncology because of its role in angiogenesis,” he added.
SF1126 is a vascular-targeted PI3 kinase inhibitor that has been shown in Phase I clinical trials to block the pAKT pathway through which angiogenic factors (VEGF) are active. “This molecule also hits a number of other targets, which is probably better for efficacy for treatment outcomes. That’s an advantage of our drug,” said Dr. Garlich.
The additional targets are all involved in processes key to cancer cell progression. These include DNA-PK (enzyme involved in DNA repair), PLK-1 (kinase involved in cell cycling for cancer growth), CK-2 (kinase that suppresses P10, nature’s brake on PI3 kinase), and PIM-1 (involved in cell cycling and division, important in lymphomas). So this molecule is designed as a dual inhibitor of angiogenesis and cell proliferation.
“This is a unique profile of inhibition, and as long as toxicity is tolerable, the hope is that hitting many aspects of cancer cells will have maximum efficacy,” Dr. Garlich noted.
There are currently two Phase I trials with SF1126—one in solid tumors and one in multiple myeloma. The company was awarded research funding by the Multiple Myeloma Research Foundation, and the trial is being conducted at four different centers.
“I think PI3 kinase is the big shining star —the one to hit for the best impact—but even beyond that, I think the next step up will be treatments that eradicate cancer stem cells,” summarized Dr. Garlich.
Controlling Feedback Loops
Mining its library of four million compounds, Exelixis has developed several potential cancer compounds. Two compounds, XL147 and XL765, target the PI3K pathway. “This is arguably the most disregulated pathway in all of human tumor biology,” said Michael Morrissey, Ph.D, president of R&D.
The reason for focusing on PI3K, he added, is that there are a number of feedback loops that emanate from m-TOR and downstream kinases that circle back to the top of the pathway and turn on AKT, an enzyme that drives tumor survival signals.
“So by inhibiting mTOR by itself, what’s been shown preclinically and now clinically, is that you actually turn on a part of this pathway that can lead to enhanced tumor survival. This is why XL765 inhibits both mTOR and PI3K,” added Dr. Morrissey.
In vivo properties of the two compounds have been optimized and have shown good exposure after oral dosing, and compelling inhibition of the targets and the pathway in tumors in preclinical models after a single oral dose. In addition, Dr. Morrissey said that a wide variety of xenograft efficacy models showed arrested tumor growth or tumor suppression by dosing the compounds in various tumor types that had either PI3k alpha mutations to activate the tumor or Ras mutations.
Phase I clinical data has shown on-target activity for both compounds, and the company is moving into Phase II studies. “The key question as we move forward is whether PI3k specific inhibition is less toxic. We see good tolerability with both compounds, but see a little more toxicity with XL765 than with XL147. We’re not sure if it’s scaffold activity,” pointed out Dr. Morrissey.
Phase II studies will be enriching for tumor types with known mutations in a pathway or alterations in a broad pathway. “We’re moving in a broad fashion to include all kinds of tumors, but at the same time, being careful to analyze the genetics and pharmacodynamics of the tumors.”
PI3K Isoform Inhibitor
Instead of focusing on a broad inhibitor of the PI3k pathway, Calistoga Pharmaceuticals has developed an isoform inhibitor called CAL-101. The isoform is p110delta, and its expression is restricted to cells of the immune system—hematopoetic origin.
“Many of the potential side effects seen with the broad-spectrum inhibitors can be overcome by selective targeting of cells of the immune system,” explained Neill Geise, Ph.D., CSO. “This also opens up the possibility of treating a range of indications including cancer, autoimmune diseases, and allergic diseases.”
Originally in-licensed from ICOS when just in early preclinical development, CAL-101 was further developed internally using Calistoga’s profiling assays.
“We’ve developed numerous assays that allow us to profile our compounds with respect to the various PI3K isoforms, and this is important for the preclinical characterization for these clinical candidates as they move into clinical development.”
A Phase I dose-escalation study has been completed, and CAL-101 is now in a second Phase I study in hematologic cancer patients (chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, and acute myeloid leukemia).
The company has a second candidate moving into clinical development later this year indicated for allergic diseases. In addition, a pipeline of earlier-stage candidates with unique isoform specificities has potential applications for a range of indications.
PI3K Signaling Studies in 3-D Models
Harvard Medical School’s department of cell biology is analyzing the regulation of cell proliferation, migration, and survival during normal morphogenesis and how they are altered in tumor genesis. Utilizing a 3-D model where cells organize into structures, which resemble the organization of cells in glandular tissues, scientists are finding that cell regulation differs in this model from that seen in a rigid petri dish.
“We’re able to significantly expand the range of biological activities that can be monitored,” said Joan Brugge, Ph.D., chair of the department. “We were interested in being able to model events that would tell us something about the architectural changes in structures that occur during tumorgenesis and then understand the signaling pathway and processes involved in that. It seemed irrelevant to study any of those processes in a monolayer.”
Her group added normal breast mammary epithelial cells in the 3-D cultures, which formed a solid mass that leads to the generation of a hollow spherical mass. The outer cells of the solid mass polarize, creating a dichotomy between the outer and inner cells, causing the inner cells to stop depositing extracellular matrix. Studies have shown that epithelial and endothelial cells have a stringent requirement for attachment to extracellular matrix in order to survive.
“So the analogy in human tissues is there are several types of early tumor lesions that can be distinguished by cells that can hyperproliferate but not fill the lumen space or lesions that are completely filled. That suggests the ability of cells to fill the lumen may be dependent on antiapoptotic activity. Now we know you need matrix signals not only for proliferation, but also for survival.”
Dr. Brugge’s lab is also focusing on how oncogenes prevent apoptosis via the PI3K and ERK pathways. “If you lose either pathway, you lose the ability of cells to protect from apoptosis.” They found that by preventing apoptosis by overexpressing BCL-2 (anti-apoptotic gene), the cells in the center of the spherical model experience stress and undergo metabolic impairment. The levels of ATP drop and the cells die. “We looked at the metabolic impairment, and the PI3K pathway was the major player. So it looks like this pathway is regulating glucose transport.”
Another discovery the group made is that when suspended mammary epithelial cells that lacked glucose were treated with anti-oxidants, the ATP levels were rescued without affecting glucose transport. “That suggests that when the cells were stressed by this nutrient deprivation (no glucose), antioxidants actually rescue the cell survival and the metabolic impairment,” explained Dr. Brugge, she added that it was not clear how this information translates into clinical applications.
Analyzing Role of PI3K Isoforms
Researchers at the Institute of Cancer at Barts and The London School of Medicine’s Center for Cell Signaling are investigating the role of PI3K isoforms in normal physiology and disease. “I thought there was something interesting about PI3k isoforms when I started in research 15 years ago,” said Bart Vanhaesebroeck, Ph.D., center lead. His group isolated the PI3K genes in various mammals and tried to use mouse knock-out models to understand gene function.
It turns out this strategy doesn’t work well for PI3K because it provides no good correlation to what inhibitors would do. Instead, Dr. Vanhaesebroeck decided to develop a model that would mimic what inhibitors do genetically.
The kinase knock-in mice have an active site with a mutation in an ATP-binding amino-acid residue, leading to inactivation of kinase. “The knock-in strategy is much more informative for drug development than the knock-out model—it’s a more real reflection of what a small molecule inhibitor will do.”
The first gene he cloned was the p110delta—one of the eight isoforms of PI3K. Expressed mainly in white blood cells, it is a good target for autoimmune diseases, allergies, inflammation, and leukemia. A p110delta PI3K knock-in mouse model was developed and worked well. Phenotypes seen in this model were more severe than in equivalent knock-out mice, “revealing some interesting biology in terms of immune signaling.” Additional research found the isoform important in controlling allergic reactions. “This biological work identified p110delta as one to go for in terms of drug development,” stated Dr. Vanhaesebroeck.
This research process was repeated with p110 alpha knock-in mice. This isoform has been found to be extremely important in insulin and metabolic signaling. Interestingly, when inactivated, the isoform does cause some changes in metabolic control, but does not induce diabetes. Further work has shown it also affects angiogenesis and blocks blood vessel formation.
The potential of PI3K as a drug target remains to be seen as clinical trials are still in the early stages. “I think you have to separate cancer from other diseases like inflammation, arthritis, etc. In noncancers, if you choose a PI3K isoform, you can overcome the complexity of the PI3K pathway because they are quite specialized in their function. But in cancer, the pathway, in my view, is universally unregulated. I think you have to hit all eight isoforms. It’s not going to be as toxic as everyone had thought, but dose-escalation studies are ongoing and we still need to see efficacy,” he concluded.
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