BACKGROUND: Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1B studies of CFZ reported signals of clinical activity in solid tumors, including small cell lung cancer (SCLC). The aim of this study was to investigate the activity of CFZ in lung cancer models. METHODS: A diverse panel of human lung cancer cell lines and a SHP77 small cell lung cancer xenograft model were used to investigate the anti-tumor activity of CFZ. RESULTS: CFZ treatment inhibited both the constitutive proteasome and the immunoproteasome in lung cancer cell lines. CFZ had marked anti-proliferative activity in A549, H1993, H520, H460, and H1299 non-small cell lung cancer (NSCLC) cell lines, with IC₅₀ values after 96 hour exposure from <1.0 nM to 36 nM. CFZ had more variable effects in the SHP77 and DMS114 SCLC cell lines, with IC₅₀ values at 96 hours from <1 nM to 203 nM. Western blot analysis of CFZ-treated H1993 and SHP77 cells showed cleavage of poly ADP ribose polymerase (PARP) and caspase-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. In SHP77 flank xenograft tumors, CFZ monotherapy inhibited tumor growth and prolonged survival, while no additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin (CDDP). CONCLUSIONS: CFZ demonstrated anti-proliferative activity in lung cancer cell lines in vitro and resulted in a significant survival advantage in mice with SHP77 SCLC xenografts, supporting further pre-clinical and clinical investigations of CFZ in NSCLC and SCLC.

Résumé confidentiel / Confidential abstract

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Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached. Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell types, less is known regarding their contribution to cellular responses to proteasome inhibitors. Importantly, previous studies in murine models have shown that individual proteasome subtypes differ in sensitivity to specific proteasome inhibitors. Furthermore, research efforts in our laboratory and others have revealed that proteasome catalytic subunit expression levels and activity profiles are altered when human cancer cells acquire resistance to proteasome inhibitors. We therefore hypothesized that changes in the relative abundances of individual proteasome subtypes contribute to the acquired resistance of cancer cells to bortezomib and carfilzomib. A major obstacle in testing our hypothesis was a lack of chemical probes suitable for use in identifying distinct proteasome subtypes. We addressed this by developing a series of bifunctional proteasome probes capable of crosslinking specific pairs of catalytic subunits colocalized within individual proteasome complexes and compatible with immunoblotting-based detection of the crosslinked subunit pairs. We confirmed the utility of these probes in discerning the identities of individual proteasome subtypes in a multiple myeloma cell line that abundantly expresses catalytic subunits of both the constitutive proteasome and immunoproteasome. Our findings indicate that constitutive proteasomes, immunoproteasomes, and intermediate proteasomes co-exist within these cells and support conclusions drawn from previous studies in other cell types. We also established non-small cell lung cancer cell line models of acquired bortezomib and carfilzomib resistance in which to test our hypothesis. Using immunoblotting and proteasome activity assays, we discovered that changes in the expression levels and activities of individual catalytic proteasome subunits were associated with the emergence of acquired resistance to bortezomib or carfilzomib. These changes were inhibitor-dependent and persisted after the selective pressure of the inhibitor was removed. Finally, results obtained using our bifunctional proteasome probes suggest that the altered abundance of an intermediate proteasome subtype is associated with acquired proteasome inhibitor resistance. Collectively, our results provide evidence linking changes proteasome composition with acquired proteasome inhibitor resistance and support the hypothesis that such changes are involved in resistance mechanisms to these inhibitors.

<p dir="ltr">Nucleic acids are promising drug candidates as they can address diseases with few “druggable” targets. Nevertheless, nucleic acids are challenging to deliver because of their large molecular weights, dense negative charges, proinflammatory activities, and short half-lives in biological fluids. Synthetic gene carriers based on cationic polymers or lipids have been used to overcome these challenges; however, their cationic nature results in dose-limiting toxicities and accelerated removal by the filtering MPS organs after systemic administration. In the past six years, several nucleic acid-based therapeutics have been approved by the FDA, formulated as lipid nanoparticles (LNPs). Nonetheless, LNPs show extensive liver accumulation after intravenous administration and, hence, are only indicated for hepatic or local vaccine delivery applications. Therefore, there is a critical unmet need for a nanocarrier that delivers nucleic acids to the extrahepatic organs without significant toxicities. To address this need, we developed Nanosac, a deformable and non-cationic nanocarrier, to deliver siRNA to solid tumors. Deformability can improve multiple aspects of the nanoparticle biotransport, ranging from circulation time and protein corona composition to biodistribution and interactions with the target cells. Meanwhile, a non-cationic carrier avoids proinflammatory complications and rapid clearance of cationic nanoparticles. For this application, we used siRNAs targeting CD47/SIRPa and PD-l/PD-L1 immune checkpoints due to their critical roles as “don't-eat-me” and “don't-find-me” signals to immune cells, respectively, which interfere with the development of innate and adaptive antitumor immune responses.</p><p dir="ltr">In the same context of enhancing the tumor delivery of nanomedicine, we developed two formulations for the small-molecule chemo drug, carfilzomib (CFZ). A nanocrystal formulation with optimized particle size had high CFZ loading, adequate colloidal stability in circulation and better antitumor activity in mice than the FDA-approved CFZ formulation. Despite its improved efficacy, the stiff nanocrystals aggravated CFZ immunotoxicity due to its excessive accumulation in mice spleens. To address this issue, we employed Nanosac technology for CFZ delivery, exploiting its deformability to reduce the non-specific spleen distribution and enhance CFZ tolerability.</p><p dir="ltr">Our results showed that Nanosac delivered siRNA to tumor cells and silenced the target protein expression better than LNPs. <i>In vivo</i>, Nanosac reduced siRNA accumulation in the MPS organs and achieved greater siRNA-mediated tumor suppression than LNPs in two murine tumor models. Moreover, Nanosac achieved greater checkpoint protein silencing in tumors, but less silencing in the MPS organs than LNPs, highlighting their differential biodistribution. The superior Nanosac performance relative to LNPs after systemic delivery is likely due to the difference in their protein coronas and cellular delivery capabilities. In addition, CFZ loading in Nanosac ameliorated CFZ immune cell toxicity <i>in vitro</i> and improved its tolerability in mice while maintaining similar therapeutic efficacy compared to the stiff nanocrystal formulation. Collectively, these findings highlight nanocarrier deformability and corona composition as viable strategies to improve the extrahepatic delivery of nucleic acids as well as to minimize toxicities related to extensive NP distribution to the off-target MPS organs.</p>