Investigating Mechanisms Determining Cancer Cell Sensitivity to Carfilzomib and Novel Strategies to Overcome Resistance

Proteasome inhibitors (PIs) are a class of FDA-approved anti-cancer agents which includes the first-generation PI bortezomib (BTZ) and second-generation carfilzomib (CFZ). Drug resistance is a major challenge in PI therapy with no solution currently available. While a few resistance mechanisms had been proposed for BTZ, little was known about CFZ resistance before the start of our studies. In this dissertation work, we investigated multiple mechanisms contributing to CFZ resistance—alterations in the drug transporter activity, metabolic stability, and proteasome activity profiles—and evaluated potential strategies to overcome CFZ resistance. We observed marked upregulation of the drug efflux transporter P-glycoprotein (P-gp) in our H23 (lung cancer) and DLD-1 (colorectal cancer) cell line models of acquired resistance. P-gp inhibition by verapamil effectively restored CFZ sensitivity in resistant cells, indicating that P-gp contributes to CFZ resistance in our model. We designed a small library of CFZ analogs lacking the pharmacophore and screened them for their abilities to reverse CFZ resistance. Our results showed that dipeptide CFZ analogs were the most effective in restoring CFZ sensitivity. This study was among the first to demonstrate the involvement of P-gp upregulation in CFZ resistance and the feasibility of using CFZ peptide analogs to reverse P-gp-mediated CFZ resistance. PI-resistant cancer cells often exhibit altered proteasome activity profiles compared to PI-sensitive cells. To further explore how these changes to the proteasome may influence cellular response to PIs, we developed a pancreatic cancer cell line model of acquired CFZ resistance. CFZ-resistant BxPC3 cells displayed a marked increase in the caspase-like (C-L) activity of the proteasome compared to parental controls. When challenged with CFZ, we also found that C-L activity was preserved in resistant cells whereas all activities were inhibited in parental cells. Using both chemical and genetic knockdown approaches, we found that co-inhibition of the C-L activity can sensitize resistant cells to CFZ. Similar effects were also observed in CFZ-resistant RPMI-8226 multiple myeloma cells. These findings suggest that enhanced C-L activity may contribute to CFZ resistance and that combined inhibition of the C-L activity may serve as a potential strategy to restore CFZ sensitivity. Since CFZ contains a tetrapeptide backbone and a highly reactive epoxyketone pharmacophore, its rapid metabolic inactivation in vivo may be a potential explanation for its lack of anti-cancer activity in solid cancers. Thus, we hypothesized that improving the metabolic stability of CFZ and its access to cancer cells may enhance its anti-cancer efficacy. Using micelle particles composed of biodegradable block copolymers poly-(ethylene glycol) (PEG) and poly-(caprolactone) (PCL), we demonstrated as a proof-of-concept that extended-release nanoformulations improved the metabolic stability and cytotoxic activity of CFZ in solid cancer cell lines. These findings supported the potential utility of polymer micelle formulations in enhancing the delivery of CFZ and improving anti-cancer efficacy CFZ against solid cancers. Findings from this dissertation work enhance our understanding of factors contributing to CFZ resistance in cancer cells. Such information may be useful for the development of next-generation proteasome inhibitors and new strategies to combat CFZ resistance in the clinic

Higher-order solutions to non-Markovian quantum dynamics via hierarchical functional derivative

Solving realistic quantum systems coupled to an environment is a challenging task. Here we develop a hierarchical functional derivative (HFD) approach for efficiently solving the non-Markovian quantum trajectories of an open quantum system embedded in a bosonic bath. An explicit expression for arbitrary order HFD equation is derived systematically. Moreover, it is found that for an analytically solvable model, this hierarchical equation naturally terminates at a given order and thus becomes exactly solvable. This HFD approach provides a systematic method to study the non-Markovian quantum dynamics of an open system coupled to a bosonic environment.Comment: 5 pages, 2 figure

Nanodelivery of a functional membrane receptor to manipulate cellular phenotype.

Modification of membrane receptor makeup is one of the most efficient ways to control input-output signals but is usually achieved by expressing DNA or RNA-encoded proteins or by using other genome-editing methods, which can be technically challenging and produce unwanted side effects. Here we develop and validate a nanodelivery approach to transfer in vitro synthesized, functional membrane receptors into the plasma membrane of living cells. Using β2-adrenergic receptor (β2AR), a prototypical G-protein coupled receptor, as an example, we demonstrated efficient incorporation of a full-length β2AR into a variety of mammalian cells, which imparts pharmacologic control over cellular signaling and affects cellular phenotype in an ex-vivo wound-healing model. Our approach for nanodelivery of functional membrane receptors expands the current toolkit for DNA and RNA-free manipulation of cellular function. We expect this approach to be readily applicable to the synthesis and nanodelivery of other types of GPCRs and membrane receptors, opening new doors for therapeutic development at the intersection between synthetic biology and nanomedicine

Dynamical invariants in non-Markovian quantum state diffusion equation

We find dynamical invariants for open quantum systems described by the non-Markovian quantum state diffusion (QSD) equation. In stark contrast to closed systems where the dynamical invariant can be identical to the system density operator, these dynamical invariants no longer share the equation of motion for the density operator. Moreover, the invariants obtained with from bi-orthonormal basis can be used to render an exact solution to the QSD equation and the corresponding non-Markovian dynamics without using master equations or numerical simulations. Significantly we show that we can apply these dynamic invariants to reverse-engineering a Hamiltonian that is capable of driving the system to the target state, providing a novel way to design control strategy for open quantum systems.Comment: 6 pages, 2 figure