Experimental Therapeutics Program
Recent accomplishments of the 3DT Program include:
- Identification of the structural determinants of the BRCA1 : estrogen receptor interaction (Ma et al., Oncogene, 2005)Â
- Developing and completing preclinical testing of a novel tumor targeting delivery system to enhance the action of tubulin depolymerizing agents (Hwang et al., Mol Cancer Ther, 2008)
- Phase I to Phase III development of CEA cancer vaccine (Marshall et al., JCO, 2005)
Additionally, there are many ongoing studies:
- RNAi Screen Identifying the Tumor Determinants That Promote Resistance to Epidermal Growth Factor Receptor-Targeted Therapy with Antibodies and Small Molecule Inhibitors
- Targeting Natural Killer Cells
- Targeting the Transcription Factor EWS/FLI1 as a Treatment for Ewing's Sarcoma
- Targeting Voltage Gated Sodium Channels in Prostate Cancer
- Targeting Nuclear Receptor (Vitamin D)/Beta-Catenin Interactions
- Therapeutic Cancer Vaccines as Therapy
- Hematopoietic Stem Cell (HSC) Transplantation and Unrelated Donor/Cord Blood Registries
- Targeting Apoptotic Pathways
- Tumor Targeting Nanocapsule Delivery of Wildtype p53 for Gene Therapy of Cancer
- Activation of the PPAR Gamma Receptor as Anti-Cancer Therapy
- Pharmacogenomics and Use of EGFR Therapies
- Understanding Complex Drug-Drug Interactions in Cancer
RNAi Screen Identifying the Tumor Determinants That Promote Resistance to Epidermal Growth Factor Receptor-Targeted Therapy with Antibodies and Small Molecule Inhibitors
Investigators: Weiner, Dakshanamurthy, Jablonski.
Hyperactivity of a central EGFR-RAS-MAPK signaling axis is essential for the growth, metastasis, and drug resistance of many cancers. Cetuximab, erlotinib and other inhibitors of EGFR signaling have emerged as modestly useful anti-cancer agents, but the efficacy of these agents is significantly limited by incompletely understood mechanisms of drug resistance. New approaches that target and circumvent these resistance mechanisms are urgently required. Dr. Weiner and colleagues hypothesized that additional changes (either genetic mutations or epigenetic modifications influencing gene expression) provide alternative signaling routes to downstream essential EGFR signaling targets. siRNA screening focused on genes functionally linked to the EGFR signaling pathway can identify tumor-intrinsic genes that regulate the tumor cell response to antigen engagement by monoclonal antibodies and small molecule inhibitors targeting EGFR. A customized 638-element siRNA library containing genes known to functionally interact with EGFR (the EGFR functional network) was developed and applied. Using this library, a restricted number of genes whose knockdown selectively alters tumor cell viability in the presence of erlotinib and other EGFR pathway inhibitors were identified. "Clusters" of these genes are known to act together in discrete sub-pathways, which are predicted to be important for regulation of EGFR-directed signaling inhibition. Most importantly, the EGFR therapy resistance network differs significantly in cell lines harboring an activating KRAS mutation as compared with cell line with wild-type amplified EGFR but wild-type KRAS. These results suggest that malignant cells with activating KRAS mutations employ distinct and potentially targetable resistance mechanisms.
Iterative screens of the EGFR interactome have revealed genes whose knockdown sensitizes two cell lines, A431 and HCT116 for loss of viability induced by either camptothecin or erlotinib; the hit profiles of two cell lines (82 for A431; 60 for HCT116) contain relatively little. These hits encode ligands, cell surface proteins, Ras adapter proteins, integrins, cytoskeletal proteins, transcription factors, phosphatases, intracellular kinases and canonical EGFR pathway components. Drugs against one such hit, aurora kinase A, sensitize HCT116 cells to erlotinib or cetuximab (not shown). This approach has identified new targets for drug development, and has prioritized established targets (i.e., aurora kinase A) for development in conjunction with EGFR pathway inhibitors. Further exploration of the validated hits is forming the basis for a 3DT-led effort to clinically validate the impact of these drug-resistance mediators on clinical response to EGFR pathway inhibitor therapy in clinical trials.
Natural killer (NK) cells attack malignant or virally infected cells. Their activation state depends on the ligation of a number of inhibitory and stimulatory receptors, such as the killer cell immunoglobulin-like receptors (KIR). Some KIR are inhibitory, detecting self HLA ligands; other KIR are stimulatory with as yet undefined ligands. It is thought that NK cells adoptively transferred to patients may selectively destroy malignant cells. These patients must lack ligands for donor inhibitory KIR receptors and have malignancies which activate NK killing. A clinical trial to evaluate and enhance the role of NK cells in the treatment of myeloid leukemias is planned by Dr. El-Shami, a new junior faculty member. Complementary laboratory studies by Dr. Hurley focus on the role of cytokines in the trafficking and surface stability of stimulatory KIR to enhance NK killing in adaptive transfer clinical trials.
The group's foundational studies have focused on defining KIR diversity in US populations and on determining the functional impact of nucleotide polymorphisms, and in the process developing reagents and reference cells for KIR identification. Drs. Hurley and Ng have just completed a study of allelic diversity in European Americans (Hou et al. 2008; Mulrooney et al. 2008; Belle et al, 2008) and are in the midst of characterizing a panel of 100 random African Americans. In a collaboration between Dr. Hurley and Dr. Dakshananmurthy, parallel functional studies have focused on stimulatory KIR and the team has identified one locus (KIR2DS3) and two alleles at other loci that do not appear to express cell-surface receptors (Steiner et al, 2008; VandenBussche et al. 2006). The Lombardi Microscopy and Imaging Shared Resource was used for the intracellular localization of KIR2DS3. Molecular dynamic simulations carried out by Dr. Dakshananmurthy aided Hurley in interpreting the impact of specific amino acid changes in causing the intracellular retention of one of the stimulatory KIR. Dakshanamurthy and Weiner are collaborating to create and test small molecule inhibitors of KIR: MHC Class I interactions to overcome inhibitory self-recognitiion and potentiate antibody-dependent cellular cytotoxicity.
Ewing's sarcoma is the result of a translocation between chromosomes 11 and 22, which fuses the EWS gene of chromosome 22 to the FLI1 gene of chromosome 11. Ewing sarcoma presents in childhood or early adulthood and is more common in males, with a peak between 10 and 20 years of age. This tumor occurs most commonly in the pelvis and proximal long tubular bones and approximately 30% are overtly metastatic at presentation. Long-term survival for metastatic disease may be less than 10%. Thus, new therapeutics are urgently needed.
The Toretsky laboratory screened a library of compounds from the NIH diversity set and identified a lead compound that directly bound to EWS-FLI1 using surface plasmon resonance. Yali Kong synthesized the lead compound YK-4-279, a derivative NSC63543 from the screening and a series of enhanced potency variants. YK-4-279 was 10-fold less effective at inhibiting non-transformed HEK293 cells that did not contain EWS-FLI1. YK-4-279 also effectively blocks RHA from binding to EWS-FLI1 in ESFT cells treated for 18 hours with increasing doses of YK-4-279 or control.
YK-4-279 also reduced ESFT xenograft growth. ESFT cells were injected orthotopically into the gastrocnemius muscle of mice (n = 15), beginning 3 days after tumor injection, five mice per treatment group were treated 3 times per week with either YK-4-279 at 1.5 mg/dose, 3.0 mg/dose, or with DMSO. The increased dose of YK-4-279 reduced the growth of the tumors without evident toxicity to the mice. These studies exemplify the tight interactions among 3DT members across the spectrum ranging from target identification through drug discovery and drug development. Here, one set of program members (Toretsky, with Uren) identified a novel target of high cancer relevance and then isolate hits from a chemical diversity library. Collaborations in 3DT (Brown, Kong and Dakshanamurthy) resulted in the successful optimization, synthesis and evaluation of in vivo activity and toxicity (Uren, Biochemistry, 2004; Toretsky, Cancer Res, 2006; Beauchamp, J Biol Chem, 2009; Erkizan et al., Nature Medicine, in press). This work exemplifies the capacity of 3DT program investigators to seamlessly bridge the gap between the identification of an exciting molecular target and the development of inhibitors with clinical potential.
Prostate cancer (PCa) is the most common cancer in men, other than non-melanoma skin cancer. While early detection and treatment has increased survival and clinical outcomes, most cases of prostate cancer are clinically silent but patients presenting with a moderate grade cancer (e.g., grade 7) are very likely to progress. These patients have high risk for developing metastatic, currently incurable prostate cancer.
Dr. Brown et al examined 17 normal and 160 malignant prostate tissue specimens categorized by Gleason score and by immunohistochemical analysis using a polyclonal (pAb) anti-Nav 1.8 antibody. Significant correlations were found between protein expression and Gleason score.
ICM-I-136 and other analogues were designed and synthesized by Dr. Brown (Anderson J, Molecular Cancer Therapeutics 2003). ICM-I-136 functionally inhibits the Nav1.8 channel and human prostate cancer cell proliferation and migration. When evaluated for in vitro and preclinical toxicity, ICM-I-136 was found to be non-toxic at 5 and 10 mg/kg. Mice with PC3 xenografts were treated with 5 or 10 mg/kg of ICM-I-136 ip once a day every other day over 15 days, with a statistically significant reduction in tumor volume after day 5 (two doses of ICM-I-136) at 10 mg/kg. These preliminary in vivo results demonstrate a significant separation of toxicity and efficacy. These studies demonstrate the ability of 3DT Program expertise to translate from basic science concepts through pre-clinical studies and to develop new agents with promise to treat cancers.
Several studies that elucidated the molecular basis and biological significance of beta-catenin/nuclear receptor interactions arose from inter-programmatic collaborations involving Drs. Byers, Shah, and Dakshanamurthy. From 2003 to 2008 these collaborations built on their previous work to show that beta-catenin interacted with and influenced the activity of the retinoic acid receptor, the vitamin D receptor (VDR) and the androgen receptor. Specifically, they established the molecular basis of the interactions and, using a combination of molecular modeling and in silico drug design, identified six new vitamin D analogues which are anticipated to be beta-catenin specific (Shah et al., Mol Cell, 2006). Dakshanamurthy and Byers are developing vitamin D analogues which will only activate VDR in situations, such as colon cancer, in which beta-catenin is elevated. Dr. Pishvaian focuses his efforts on translational trials in this area.
We have contributed to understanding how immune responses can help control cancer focusing on the development of CEA-targeting pox virus vaccines. Starting in 2000 and continuing through the current funding period we have developed CEA-based vaccines from phase I singleâ€‘agent clinical trials to a phase III randomized clinical trial designed to justify vaccine licensing by the U.S. Food and Drug Administration (FDA). The speed of vaccine development has been hastened by key translational discoveries allowing for significant improvements in vaccine potency. Most recently, we have shifted our focus to less heavily treated patients with lower tumor burdens. Our current active trial compares traditional subcutaneous administration of the vaccines to ex vivo dendritic cell immune stimulation in Stage 4 colon cancer patients following resection of their metastases. The primary endpoints for this trial are immune responses and clinical benefit. This CEA vaccine work is supported by long-standing collaborations with Drs. Jeffrey Schlom (NCI) and Michael Morse (Duke).
Newer vaccine constructs and immune stimulating agents are being tested in the 3DT Program. First in human trials of vaccines targeting CEA, RAS, and novel synthetic peptides including CpG oligonucleotides are currently under way. Phase II studies of the RAS vaccines are planned in colorectal cancer for 2009. Dr. Weiner's addition to the 3DT Program offers a new scientific base for these efforts, both to provide laboratory support for immunological correlate studies and to generate new hypotheses that can be tested in the clinical setting. For example, his laboratory has shown that immune responses directed against HER2/neu are augmented by combined therapy of HER2/neu expressing syngeneic tumors growing in human HER2/neu transgenic mice (unpublished; manuscript submitted).
HSC transplantation has been used to restore hematopoiesis and to treat malignancy. Treatment complications, notably alloreactivity, have limited its applicability. Working with a team of US investigators, the National Marrow Donor Program (NMDP) and the Center for International Blood and Marrow Transplant Research (CIBMTR), Dr. Hurley has provided the project design for and oversight of the retrospective high resolution assignment of HLA alleles in hematopoietic stem cell donors and their unrelated recipients. Combined with clinical data, the HLA matching information was used to refine guidelines for optimal unrelated donor selection.
Based on these matching criteria and in order to optimize donor selection, Dr. Hurley has collaborated with investigators from the NMDP and other international registries to refine the search algorithm that matches patients with unrelated donors. The algorithm is built to predict the probability of a high resolution match based on the frequencies of HLA haplotypes in human populations. Population studies by Dr. Hurley in collaboration with Dr. Jennifer Ng have defined population substructure among European Americans; this will further refine haplotype predictions (Mack et al, 2008). Studies by Drs. Ng and Hurley to define the genetic contribution of Native Americans to the Hispanic U.S. population are underway with national and international collaborators.
The 3DT Program continues its focus on apoptosis as a target, completing a series of translational clinical trials of novel agents and moving an agent developed within the program into the clinic. Below is a summary of one of the key projects in the current grant cycle.
AKT as a Target for Inducing Apoptosis
AKT-1, the expression protein product of akt-1 proto-oncogene, plays a vital role in cancer progression by stimulating proliferation and inhibiting apoptosis of cancer cells. Glazer conducted basic and drug development studies on the role of AKT in oncogenesis. These included development of a transgenic mouse model expressing AKT1 in the mammary gland, as well as de novo drug development studies of AKT inhibitors. Glazer is presently examining the role of the AKT kinase, 3-phosphoinositide-dependent protein kinase-1 (PDK1), which may be a useful anticancer drug target (Liang et al., Mol Pharmacology, 2006).
One direct way to assess the role of AKT1 is to employ antisense oligodeoxynucleotide (AON) therapy, assuming it does not result in off-target effects. To test this, Drs. Marshall and Hwang performed a phase I trial of a novel AKT-antisense molecule, RX-0102. RX-0201 (Archexin), a 20-mer oligonucleotide with sequence complementary to akt-1 mRNA, was designed to inhibit the expression of akt-1 within cancer cells in cancer patients. Pre-clinical studies conducted on RX-0201 by Dr. Glazer (BC) demonstrated that RX-0201 has significant in vitro and in vivo anti-cancer activity with a favorable safety profile. The objectives of the phase I trial were to determine the maximum tolerated dose (MTD) and to establish safety and pharmacokinetic (PK) profiles of RX-0201 in patients with an advanced cancer. RX-0201 was administered to patients with an advanced cancer by up to 2 cycles of continuous intravenous 14-day infusions using a rapid dose escalation until at least a grade (G) 2 toxicity was observed, followed by a traditional dose escalation. 17 cancer patients were treated at doses ranging from 6 to 315 mg/m2/d. The MTD was 315 mg/m2/d based upon compound-related grade 3 fatigue in two patients at that dose; no other significant, compound-related, adverse events were observed. Limited clinical activity was observed. 250 mg/m2/d is being used as the dose for the follow-on phase IIa trial in renal cancer. Tumor samples were obtained as part of the trial to demonstrate on-target effects of treatment; these are being evaluated for alterations in the AKT pathway. (Malik et al., ASCO, 2006)
Drs. Chang and Pirollo have developed a tumor-targeting nanocapsule delivery system for use in nanomedicine. These nanosize particles are composed of a cationic lipid capsule surrounding the payload, with an anti-transferrin receptor single chain antibody fragment (TfRscFv) decorating the surface to serve as the tumor targeting moiety (Xu L, Mol Cancer Ther 2002). The presence of the TfRscFv moiety bestows high levels of tumor specificity when administered through the blood stream, targeting and efficiently delivering its payload to tumor sites, including those in the brain. This is a platform technology with a variety of potential applications since these nanocapsules can be employed to deliver a variety of molecules as the payload (Pirollo KF, Human Gene Ther 2006; Pirollo KF, Can Res 2007).
The first translational product in the pipeline of this platform technology is the TfRscFv-nanocapsule complex delivering the human wt-p53 gene as an anti-cancer gene therapy agent. This p53 containing nanocapsule has shown impressive anti-cancer activity in a variety of in vitro and in vivo models, both as a single agent and in combination with radiation and chemotherapy. We began the formal clinical testing of this agent in 2005 with a phase I trial initiated within the 3DT Program. Members of the 3DT Program were instrumental in the design and writing of the clinical protocols. A second Phase I trial delivering another tumor suppressor gene, RB94 (Pirollo KF, Clin Can Res 2008), will be conducted at the M.D. Anderson Cancer Center in subjects with advanced cancers.
Agonists of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARg), such as the thiazolidinediones (TZDs) have shown potential in the treatment of a variety of cancer types in vitro and in mouse models. However, the clinical efficacy of second generation PPAR gamma agonists in humans has been mixed. We have conducted basic, chemopreventative and drug development studies on the role of PPARg in mammary tumorigenesis. These included development of a transgenic mouse model expressing a dominant-negative PPARg in the mammary gland by Dr. Glazer, as well as de novo drug development studies of PPARg agonists. Preclinical studies of the PPARg agonist CS-7017, in collaboration with Daiichi-Sankyo, investigated its activity in pancreatic carcinoma and liposarcoma xenografts with a focus on biomarkers that may be predictive of drug efficacy. 3DT members Drs. Marshall and Pishvaian have conducted Phase I/II clinical trials of the PPARg agonist CS-7017 and plan combinations with bexarotene or bortezomib in patients with GI cancer to determine if activation of PPARg potentiates chemotherapy (ASCO 2008; ECCO, 2008).
In the recently completed a Phase I clinical trial with this third generation PPARg agonist, which is 50 times more potent than prior agonists, our investigators observed one partial response and broad safety. Patient samples were collected for extensive analysis of serum markers of PPAR gamma activity. Additionally, peripheral blood mononuclear cells were collected and harvested for RNA to perform quantitative RT-PCR on PPAR gamma responsive intracellular genes, such as p21 and p27. The study of PPARg antagonists has spanned the translational spectrum from basic target discovery through drug discovery, drug development and clinical investigations, all of which were conducted at Lombardi. These studies demonstrate how Lombardi investigators have developed meaningful inter-programmatic interactions that further the aims of each participating program.
Responses to, and toxicities from, chemotherapy vary substantially from person to person. While there are many sources for this inter-individual variation, genetic factors are known to account for 15-30% of the differences in drug metabolism and response. These genetic variations affect the expression and function of proteins responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs, as well as in the targets of drug therapy.
In a phase II prospective pharmacogenomics trial, patients with locally advanced Head and Neck cancer are being enrolled using their genetic variations in DNA repair pathway enzymes to determine whether they should receive cisplatin or cetuximab with concurrent radiation therapy to treat their disease. The primary objective is to determine whether using this approach can improve disease free survival measured two years after completing therapy.
Deeken and colleagues are testing the hypothesis that highly active antiretroviral therapy (HAART) can interact with anticancer chemotherapy, reducing efficacy and increasing the risk of toxicity, by inducing or inhibiting drug metabolism, respectively. Their initial study is to investigate sunitinib, an orally available multiple tyrosine kinase inhibitor of VEGFR1/2/3, Kit, and PDGFR alpha and beta. The parent drug is metabolized to an active compound SU12662 by CYP3A4, and further metabolized to an inactive metabolite, also by CYP3A4. Besides ritonavir, other HAART agents thought to inhibit CYP3A4 in different degrees include delavirdine, efavirenz, amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, and saquinavir. Nevirapine and efavirenz can induce CYP3A4, which may in turn decrease serum levels of sunitinib and reduce its efficacy. Finally, sunitinib could alter the absorption and/or the elimination of many of these drugs in patients with HIV. Dr. Deeken (protocol chairman), working with Drs. Little (NCI), Dezube (Beth Israel/Harvard), and the AIDS Malignancy Consortium, supported by the NCI U01 Phase I network, is performing a clinical trial of sunitinib in HIV patients receiving HAART therapy focused on pharmacokinetic correlates. This pilot study should be the first of many such studies supported by the newly formed NCI Working Group for PK Assessment of Antiretroviral Therapy and Novel Cancer Therapeutics, with the AMC serving as the lead cooperative group.