Preclinical and Clinical Imaging and Treatment of Multiple Myeloma with CMYC-MAX Nanoparticles:
Multiple myeloma (MM) is a malignancy derived from a clone of plasma cells, the terminally differentiated B-lymphocytes responsible for antibody production. MM is the second most common hematologic malignancy in the United States, and accounts for 1% of cancer deaths. Despite recent advances, the 5-year survival rate in patients with MM is less than 40%. The malignant cells in MM respond well to several classes of chemotherapy, (e.g. proteasome inhibitors, immunomodulatory drugs (IMiDs), and alkylating agents), which have increased the median survival from 3 years to over 6 years. Unfortunately, in virtually all patients, the remissions induced by the best current chemotherapies are transient, and patients eventually relapse and die from progressive disease. Innovative approaches to myeloma treatment are urgently needed.
Transcription factors’ relative position downstream as integrators of multiple signaling cascades makes them an attractive therapeutic target with convergence of several signaling pathways, perhaps decreasing the risk of therapy-resistance that can occur with cytoplasmic signaling inhibitors by up-regulation of parallel signaling pathways. The b-HLHZIP transcription factor c-Myc (MYC) is a powerful oncogene activated in many types of cancer. MYC activation is increasingly recognized as a common transforming event in myeloma. Therefore, targeting MYC either directly, or indirectly, may be a useful therapeutic approach in MM. However, MYC remains a challenging target for drug discovery due to the difficulty of inhibiting protein-protein or protein-DNA interactions with small molecules. Unfortunately, several effective small-molecule inhibitors of the MYC-MAX interaction in vitro failed when applied in vivo due to rapid systemic metabolism, poor bioavailability, and an inability to achieve effective drug levels in tumors. We have demonstrated substantial increased survival in an aggressive metastatic mouse model of MM by delivering a novel Sn 2 lipase labile Myc-Max inhibitor prodrug (MI1-PD) with lipid-based nanotherapeutics targeted to the VLA-4 integrin receptor. The overarching aim of project 1 is to develop VLA-4 targeted cMyc-PD nanotherapeutics that alone or in combination with chemotherapy maximize metastatic MM survival in mice.
This multi-PI proposal affords an interdisciplinary collaboration that integrates the nanomedicine expertise of Dr. Gregory Lanza, MD PhD (PD/ PI), Professor of Medicine and the co-Director of the Consortium for Translation Research in Advanced Imaging and Nanomedicine (C-TRAIN) with Dr. Michael Tomasson MD (PI), a board certified Hematologist, Associate Professor of Medicine and Scientific Director of the Multiple Myeloma Program at the Siteman Cancer Center at Washington University School of Medicine. Additional co-investigators include Steven Fletcher, Ph.D., an Assistant Professor at the University of Maryland School of Pharmacy, who has designed, synthesized, and published numerous highly potent cMyc-Max dimerization inhibitors in conjunction with Dr. Edward V. Prochownik, MD PhD, Professor of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine and Director of Oncology research, Childrens Hospital of Pittsburgh who is internationally recognized for the characterization of cMYC-MAX antagonists
We have demonstrated substantial increased survival in an aggressive metastatic mouse model of MM by delivering a novel Sn 2 lipase labile Myc-Max inhibitor prodrug (MI1-PD) with lipid-based nanotherapeutics targeted to the VLA-4 integrin receptor. Second and third generation cMYC-MAX inhibitors have been synthesized and modified into new prodrugs. In vitro screening studies in human cancer cells have demonstrated that the new compounds have markedly greater bioactivity than the original inhibitor, MI1-PD.
Preliminary results from n vivo testing of the new prodrugs in a VLA-targeted micelles in disseminated multiple myeloma in mice confirmed the previous finding with MI1-PD by extending animal survival twice the length of the controls. Lack of expected activity in the new compounds in mice suggest that the drugs are species specific. In parallel studies in mouse and human myeloma cells are ongoing to assess species specificity. This possible conclusion is supported by the knowledge that both new compounds bind cMYC spatially differently from MI1-PD and by robustly confirmed activity in human melanoma cells. Further work is ongoing to conclude the ongoing mouse study to allow firm conclusions and to restart a new animal study using human cells.
Communication of this novel approach to cMYC antagonism with VLA-4 targeted micelles and Sn2 prodrug inhibitors were published in two online digital subscriber serials circulating to corporate, government, media, and other nontraditional scientific consumers bases. Adjacent Government: https://www.adjacentgovernment.co.uk/research-science-innovation-
news/nanotherapy-role-multiple-myeloma-treatment/27336/ and International Innovation: https://www.internationalinnovation.com/a-new-nanotherapy-for-multiple-myeloma/.
Additionally, presentations related to this line of research have been made at invited presentations over the last 4 months to the 14th European Symposium on Controlled Drug Delivery (Denmark), The Chinese American Society of Nanotechnology and Nanomedicine (Beijing), and the Gordon Research Conference on Medical Drug Delivery (Waterville Valley).
The initial US patent for this prodrug technology has been approved and will issue in the next few weeks. A continuous submission strategy for additional claims are proceeding that further expand the breadth of this patent, but were to broad to pursue initially.
Interest in licensing of the technology has been expressed by a European group while plans to move forward with a US biotechnology start-up are in the discussion/planning stages with a corporate entity, pending further product candidate definition.
Our immune defense system naturally activates B-lymphocytes to plasma cells when stimulated by foreign antigens to produce protective antibodies. Occasionally, a normal plasma cell will accumulate mutations causing it to proliferate out of control in the bone marrow, over-secrete a single abnormal antibody or M-protein and damage normal tissues. This is multiple myeloma (MM). MM responds well to current chemotherapies but virtually all patients experience relapses with transient remissions and most eventually die of their disease. The genetic architecture of MM is complex, but a common feature of disease progression is activation of an oncogene known as c-Myc (MYC).
MYC is a helix-loop-helix transcription factor that binds as a heterodimer to DNA and regulates many critical cellular functions, including proliferation, differentiation, and apoptosis. MYC over-expression is associated with many cancer including MM. MYC overexpressing cancer cells become “oncogene addicted” and require constant MYC-MAX DNA stimulation to survive. Disrupting this pathway by interfering with MYC-MAX complex formation or MYC-MAX binding to DNA can promote MM cell death. (1-3) Hence, effective inhibitors of MYC-MAX dimer formation have been consider a holy grail of cancer therapy.
Unlike enzymes and cell surface receptors that have ligand binding pockets that have proven to be good drug targets, the transcription factors MAX and MYC when bound to DNA have no such sites and their interaction domain is relatively flat and featurelesst. Without a clear ligand-binding site to target, these transcription factors were considered pharmaceutically undruggable, until the biochemical details of MYC-MAX formation and critical interface regions were defined. (4,5)This led to the discovery of small-molecule inhibiting drugs, such as those compounds reported by Dr. Ed Prochownik and Dr. Steven Fletcher from Children’s Hospital in Pittsburgh Pittsburgh and The University of Maryland Medical Center. (6,7) These anti-MYC drugs and others like them were effective in bench assays but had limited efficacy in animal models. While in their unmodified forms, some of these agents have shown modest in vivo effects in select tumor types, the compounds were largely destroyed during circulation in the blood and intracellular bioavailability of the inhibitors reaching the MM cells was poor.
Drs. Gregory Lanza and Michael Tomasson, members of the Siteman Cancer Center at the Washington University School of Medicine in Saint Louis, understood this dilemma and envisioned a novel nanotechnology solution. Their vision was to transform the small molecule cMyc-inhibitors into lipid prodrugs and incorporate them into minute micelles functionalized to home to a common MM cell surface marker, VLA-4.
The cMyc-inhibitor prodrug utilizes a natural cell membrane constituent, lecithin, and coupling of the drug to a modified end of the middle fatty acid (Sn2), the oily part of the lipid. (8) Incubation of the cMyc-prodrug MM cells revealed more rapid adsorption of the drug than the free inhibitor. Unlike the free drug, the cMyc-prodrug is not active as a lipid conjugate. When targeted and bound to the target cancer cell, the lipid membranes of the particle and cell fuse, allowing the MYC-prodrug to transfer into the outer cell membrane leaflet and then “flip” into the inner membrane leaflet, which is contiguous with all of the inner cell membranes. The active cMYC-inhibitor is reconstituted by cytosolic lipase enzyme degradation of the abnormal lipid prodrug at the Sn2 ester and further spontaneous release of the short carbon tail due to the mild acidic nature of the cytosol. This small molecule inhibitor, once bound to cMyc, disrupts complexation with MAX in the nucleus, and prevents binding of the oncogenic dimer to DNA. The signal for continued MM proliferation is blocked and the MM cells, addicted to the cMyc-Max stimulation, die.
Another key advancement in this therapeutic strategy was the development of tiny polysorbate/phospholipid-based nanoparticles called micelles, which are similar in size to an antibody. (8) This minute size allows the micelles to penetrate rapidly into bone marrow, spleen, and other extravascular MM metastatic depots. They effectively target MM via a VLA-4 peptidomimetic that recognizes the activated MM cell surface receptor. The VLA-4-cMyc-prodrug micelles pass through normal tissues and cells rapidly due to their small amorphous size but bind and fuse irreversibly to MM cells presenting activated VLA-4. This unique fusion of the micelle lipid surface with the MM cell membrane surface initiates the drug transfer and a “kiss of death”. This novel drug delivery mechanism is termed “contact facilitated drug delivery” (CFDD).
VLA-4-cMyc-prodrug micelle was demonstrated in vivo using disseminated mouse model of MM, known as 5TGM1/ KaLwRij. (9,10) In contradistinction to nontargeted drug laden micelles, targeted drug free micelles, equivalent free MYC-inhibitor or MYC-inhibitor prodrug injected systemically, mice that received the VLA-4-cMyc-prodrug micelle system survived nearly twice as long. The results were definitive.
This CMMN program continues on a translational track transforming the best candidate MYC-inhibitors developed by Drs. Prochownik and Fletcher into lipid prodrugs and evaluating them in vitro and in survival animal models using the micelle delivery approach. Efforts are underway to identify the best of Myc-inhibitor prodrug candidate for translation by licensure and new company formation in 2017. Particularly promising compounds include analogs of some of originally described anti-Myc drugs that have greatly improved intracellular half-lives.
- Prathapam T, Aleshin A, Guan Y, Gray JW, Martin GS. p27Kip1 mediates addiction of ovarian cancer cells to MYCC (c-MYC) and their dependence on MYC paralogs. J Biol Chem 2010;285:32529-38.
- Choi PS, van Riggelen J, Gentles AJ et al. Lymphomas that recur after MYC suppression continue to exhibit oncogene addiction. Proc Natl Acad Sci U S A 2011;108:17432-7.
- Holien T, Vatsveen TK, Hella H, Waage A, Sundan A. Addiction to c-MYC in multiple myeloma. Blood 2012;120:2450-3.
- Sauvé S, Tremblay LN, Lavigne P. The NMR solution structure of a mutant of the Max b/HLH/LZ free of DNA: insights into the specific and reversible DNA binding mechanism of dimeric transcription factors. J Mol Biol 2004;342:813-32.
- Lavigne P, Crump M, Gagné S, Hodges R, Kay C, Sykes B. Insights into the mechanism of heterodimerization from the 1H-NMR solution structure of the c-Myc-Max heterodimeric leucine zipper. J Mol Biol 1998;281:165-81.
- Mustata G, Follis A, Hammoudeh D et al. Discovery of novel Myc-Max heterodimer disruptors with a three-dimensional pharmacophore model. J Med Chem 2009;52:1247–1250.
- Yin X, Giap C, Lazo JS, Prochownik EV. Low molecular weight inhibitors of Myc-Max interaction and function. Oncogene 2003;22:6151-9.
- Pan D, Pham CT, Weilbaecher KN, Tomasson MH, Wickline SA, Lanza GM. Contact-facilitated drug delivery with Sn2 lipase labile prodrugs optimize targeted lipid nanoparticle drug delivery. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2016;8:85–106.
- Pan D, Kim B, Hu G et al. A strategy for combating melanoma with oncogenic c-Myc inhibitors and targeted nanotherapy. Nanomedicine (Lond) 2015;10:241-51.
- Soodgupta D, Pan D, Cui G et al. Small molecule MYC inhibitor conjugated to integrin-targeted nanoparticles extends survival in a mouse model of disseminated multiple myeloma. Mol Cancer Ther 2015;14:1286-94.