Development of constitutively synergistic nanoformulations to enhance chemosensitivity in T-cell leukemia.

Advances in multiagent chemotherapy have led to recent improvements in survival for patients with acute lymphoblastic leukemia (ALL); however, a significant fraction do not respond to frontline chemotherapy or later relapse with recurrent disease, after which long-term survival rates remain low. To develop new, effective treatment options for these patients, we conducted a series of high-throughput combination drug screens to identify chemotherapies that synergize in a lineage-specific manner with MRX-2843, a small molecule dual MERTK and FLT3 kinase inhibitor currently in clinical testing for treatment of relapsed/refractory leukemias and solid tumors. Using experimental and computational approaches, we found that MRX-2843 synergized strongly-and in a ratio-dependent manner-with vincristine to inhibit both B-ALL and T-ALL cell line expansion. Based on these findings, we developed multiagent lipid nanoparticle formulations of these drugs that not only delivered defined drug ratios intracellularly in T-ALL, but also improved anti-leukemia activity following drug encapsulation. Synergistic and additive interactions were recapitulated in primary T-ALL patient samples treated with MRX-2843 and vincristine nanoparticle formulations, suggesting their clinical relevance. Moreover, the nanoparticle formulations reduced disease burden and prolonged survival in an orthotopic murine xenograft model of early thymic precursor T-ALL (ETP-ALL), with both agents contributing to therapeutic activity in a dose-dependent manner. In contrast, nanoparticles containing MRX-2843 alone were ineffective in this model. Thus, MRX-2843 increased the sensitivity of ETP-ALL cells to vincristine in vivo. In this context, the additive particles, containing a higher dose of MRX-2843, provided more effective disease control than the synergistic particles. In contrast, particles containing an even higher, antagonistic ratio of MRX-2843 and vincristine were less effective. Thus, both the drug dose and the ratio-dependent interaction between MRX-2843 and vincristine significantly impacted therapeutic activity in vivo. Together, these findings present a systematic approach to high-throughput combination drug screening and multiagent drug delivery that maximizes the therapeutic potential of combined MRX-2843 and vincristine in T-ALL and describe a novel translational agent that could be used to enhance therapeutic responses to vincristine in patients with T-ALL. This broadly generalizable approach could also be applied to develop other constitutively synergistic combination products for the treatment of cancer and other diseases.

Coxsackievirus B3 infects and disrupts human induced-pluripotent stem cell derived brain-like endothelial cells.

Coxsackievirus B3 (CVB3) is a significant human pathogen that is commonly found worldwide. CVB3 among other enteroviruses, are the leading causes of aseptic meningo-encephalitis which can be fatal especially in young children. How the virus gains access to the brain is poorly-understood, and the host-virus interactions that occur at the blood-brain barrier (BBB) is even less-characterized. The BBB is a highly specialized biological barrier consisting primarily of brain endothelial cells which possess unique barrier properties and facilitate the passage of nutrients into the brain while restricting access to toxins and pathogens including viruses. To determine the effects of CVB3 infection on the BBB, we utilized a model of human induced-pluripotent stem cell-derived brain-like endothelial cells (iBECs) to ascertain if CVB3 infection may alter barrier cell function and overall survival. In this study, we determined that these iBECs indeed are susceptible to CVB3 infection and release high titers of extracellular virus. We also determined that infected iBECs maintain high transendothelial electrical resistance (TEER) during early infection despite possessing high viral load. TEER progressively declines at later stages of infection. Interestingly, despite the high viral burden and TEER disruptions at later timepoints, infected iBEC monolayers remain intact, indicating a low degree of late-stage virally-mediated cell death, which may contribute to prolonged viral shedding. We had previously reported that CVB3 infections rely on the activation of transient receptor vanilloid potential 1 (TRPV1) and found that inhibiting TRPV1 activity with SB-366791 significantly limited CVB3 infection of HeLa cervical cancer cells. Similarly in this study, we observed that treating iBECs with SB-366791 significantly reduced CVB3 infection, which suggests that not only can this drug potentially limit viral entry into the brain, but also demonstrates that this infection model could be a valuable platform for testing antiviral treatments of neurotropic viruses. In all, our findings elucidate the unique effects of CVB3 infection on the BBB and shed light on potential mechanisms by which the virus can initiate infections in the brain.

Group B Streptococcus-Induced Macropinocytosis Contributes to Bacterial Invasion of Brain Endothelial Cells.

Bacterial meningitis is defined as serious inflammation of the central nervous system (CNS) in which bacteria infect the blood-brain barrier (BBB), a network of highly specialized brain endothelial cells (BECs). Dysfunction of the BBB is a hallmark of bacterial meningitis. Group B Streptococcus (GBS) is one of the leading organisms that cause bacterial meningitis, especially in neonates. Macropinocytosis is an actin-dependent form of endocytosis that is also tightly regulated at the BBB. Previous studies have shown that inhibition of actin-dependent processes decreases bacterial invasion, suggesting that pathogens can utilize macropinocytotic pathways for invasion. The purpose of this project is to study the factors that lead to dysfunction of the BBB. We demonstrate that infection with GBS increases rates of endocytosis in BECs. We identified a potential pathway, PLC-PKC-Nox2, in BECs that contributes to macropinocytosis regulation. Here we demonstrate that downstream inhibition of PLC, PKC, or Nox2 significantly blocks GBS invasion of BECs. Additionally, we show that pharmacological activation of PKC can turn on macropinocytosis and increase bacterial invasion of nonpathogenic yet genetically similar Lactococcus lactis. Our results suggest that GBS activates BEC signaling pathways that increase rates of macropinocytosis and subsequently the invasion of GBS.