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

Abstract

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.

Keywords: Combination therapy; Drug delivery; Drug screening; Lipid nanoparticle; Nanotechnology; Synergy.

Figure 1.. Combined MRX-2843 and vincristine synergize to inhibit T-ALL cell growth in high-throughput combination drug screens.

a) Illustration of a ratiometric drug screen combining the dual MERTK/FLT3 inhibitor, MRX-2843, with methotrexate and/or vincristine in a panel of 13 B- and T-ALL cell lines as measured by luminescent viability assay (72 h, Z’≥0.5). b) Single-agent dose response curves and (c) lineage-specific frequencies of combined drug synergy, additivity, or antagonism for 537 unique pairwise or triplet drug combinations as measured in parallel and assessed via the Bliss Independence model. d) Scatter plots of mean drug synergy as a function of mean growth inhibition grouped by cell lineage. Ratiometric drug synergy is conserved across T- and ETP-ALL cell lines with synergy among distinct molar ratios of MRX-2843 and vincristine. e) Drug synergy and antagonism appear at distinct doses and molar ratios in cells from the T- and ETP-ALL lineage. (c-e) Synergy represents percent reduction in cell density greater or lesser than that predicted by the Bliss Independence model with synergy (>1%) and antagonism (<−1%). (e) Sphere size and saturation correspond to the mean magnitude of synergy observed across T- and ETP-ALL cell lines (n=6) for each pairwise and triplet drug combination among 537 tested.

Figure 2.. In silico and in vitromodels prioritize pairwise drug synergy between MRX-2843 and vincristine among screened combinations.

a) Comparison of higher order (3-drug) and lower order (2-drug) synergy models to high-throughput combination drug screening data indicates that experimentally observed drug synergy is predominantly attributable to pairwise drug interactions. b) Comparison of expected (dashed) and observed (solid) Jurkat (T-ALL) cell viability following exposure to continuous pairwise drug gradients demonstrates that MRX-2843 and vincristine are most consistently synergistic. (a) Data points show 378 triplet drug combination responses measured in T- and ETP-ALL cell lines (n=6) via HTS. Data in (b) represent mean cell viability (72 h) as measured by nuclear dye exclusion.

Figure 3.. Lipid nanoparticles co-encapsulating MRX-2843 and vincristine constitutively maintain defined drug ratios following co-formulation and intracellular delivery.

a) Illustration of a pH gradient-based method of drug co-loading that relies upon (b) pH-dependent MRX-2843 and vincristine lipophilicity and charge. c) A plot of mean drug synergy versus MRX-2843:vincristine mole ratio defines synergistic (8.9 – 90), additive (90 – 160), and antagonistic (≥160) drug ratios. d) T-ALL tailored combination drug formulations encapsulating synergistic (Syn), additive (Add), and antagonistic (Antag) drug ratios shown at nanometer-scale size and uniformity as measured by dynamic light scattering and (inset) transmission electron microscopy e) Drug co-loading as measured by LC-MS. f) Intracellular drug delivery kinetics following treatment of LOUCY (ETP-ALL) cells with Antag nanoparticles over 24 h as measured by LC-MS. g) Comparison of growth inhibition following treatment with Syn nanoparticles, as well as combined and free MRX-2843/vincristine demonstrates that nanoparticle encapsulation further enhances in vitro drug synergy as measured by luminescent cell viability assay in Jurkat (T-ALL) cells (72 h) and (h) LOUCY (ETP-ALL) (72 h). (c) The line traces the trend in mean response between tested ratios, and hashes delineate the range of +/− 1% synergy, which we define as additivity. (d) Scale bars measure 80 nm, and PDI stands for polydispersity index. (e) Data points represent drug ratios from distinct nanoparticles batches as measured by LC-MS. Best fit by linear regression is shown with coefficient of linearity. (f) Vertices are mean values ± SD of intracellular drug concentrations of 3 replicates per time point as measured by LC-MS. (g,h) Individual values (dots) and mean values ± SD of 3 replicates from a single experiment are shown, where the dashed line represents the expected effect for an additive interaction (GI_exp). Mean differences were evaluated by one-way ANOVA with Tukey’s correction for multiple comparisons, where * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 4.. Comparative potency of ratiometric drug formulations in primary T-ALL samples ex vivo.

a) Approach to testing ratiometric nanoformulations in T-ALL or near ETP-ALL samples obtained from patient blood or bone marrow (BM) at initial diagnosis. b) Expected (Exp.; dashed) and observed (Obs.; solid) dose-dependent growth inhibition from synergistic and additive ratiometric drug formulations as measured by luminescent viability assay (96 h). c) Heatmap of GI₅₀s and P values from (c) Syn and (d) Add drug formulations shown in (b). (b) Mean values ± SD of growth inhibition scaled against the concomitant MRX-2843 doses in the nanoparticles. Exp. dose response curves in (b) were calculated with the Bliss Independence model of drug synergy between single-agent liposomal MRX and vincristine free drug dose responses. (c,d) Heatmaps show GI50 ± standard error (nM) of Syn and Add potencies scaled by MRX-2843 dose. Bar charts show associated log transformed P values of unpaired parametric t tests. Asterisks (*) denote extrapolated GI50 values. All Obs. dose responses were run in triplicate, while Exp. dose responses were run either in duplicate (1172a) or triplicate (1147a, 1369b) in a single experiment.

Figure 5.. Nanoparticles containing MRX-2843 and vincristine at an additive ratio mediate superior antileukemia activity in a murine ETP-ALL xenograft model.

a,b) NRG mice were inoculated with a luciferase-expressing ETP-ALL cell line (LOUCY-luc; 2 million cells). Mice were randomized to groups with equal starting disease burden (n=7–8) and treatment with synergistic (Syn) or additive (Add) liposomal formulations of MRX-2843 and vincristine or vehicle nanoparticles without drug (Veh) was initiated 20 days later. Arrows indicate treatment administration. c) Disease burden was determined at intervals by bioluminescence imaging. Data are reported as mean ± SEM. Differences were determined by two-way ANOVA. d) Differences in median survival (MS) were calculated by the log-rank method. e,f) NRG mice were inoculated intravenously with 3 million LOUCY-luc cells, then randomized to groups (n=9–10) and treatment with the indicated liposomal formulations of MRX-2843 and vincristine or Veh was initiated 20 days later. f) Differences in median survival were calculated by the log-rank method.