A cellular hierarchy framework for understanding heterogeneity and predicting drug response in acute myeloid leukemia.

The treatment landscape of acute myeloid leukemia (AML) is evolving, with promising therapies entering clinical translation, yet patient responses remain heterogeneous, and biomarkers for tailoring treatment are lacking. To understand how disease heterogeneity links with therapy response, we determined the leukemia cell hierarchy makeup from bulk transcriptomes of more than 1,000 patients through deconvolution using single-cell reference profiles of leukemia stem, progenitor and mature cell types. Leukemia hierarchy composition was associated with functional, genomic and clinical properties and converged into four overall classes, spanning Primitive, Mature, GMP and Intermediate. Critically, variation in hierarchy composition along the Primitive versus GMP or Primitive versus Mature axes were associated with response to chemotherapy or drug sensitivity profiles of targeted therapies, respectively. A seven-gene biomarker derived from the Primitive versus Mature axis was associated with response to 105 investigational drugs. Cellular hierarchy composition constitutes a novel framework for understanding disease biology and advancing precision medicine in AML.

Development of a Rapidly Dissolvable Oral Pediatric Formulation for Mefloquine Using Liposomes.

Mefloquine (Mef), a poorly soluble and highly bitter drug, has been used for malaria prophylaxis and treatment. The dosage form for Mef is mostly available as adult tablets, and thus children under the age of 5 suffer from poor medication adherence. We have developed a stable, rapidly dissolvable, and palatable pediatric formulation for Mef using liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol with a mean diameter of ∼110 nm. Mef was actively loaded into the liposomes via an ammonium sulfate gradient using the solvent-assisted loading technology (SALT) developed in our lab. Complete loading of Mef inside the liposomal core was achieved at a high drug-to-lipid ratio (D/L) of 0.1-0.2 (w/w), and the final drug content in the formulation was ∼8 mg/mL, well above the solubility of Mef (<0.6 mg/mL in simulated fluids). The strong bitterness of Mef was masked by the liposomal encapsulation as measured by an electronic tongue. Incubating the Mef-liposomes (Mef-Lipo) in the simulated gastric fluid (pH 1.2) and the simulated intestinal fluid containing 3 mM sodium taurocholate (pH 6.8) induced changes in liposome size and the polydispersity, resulting in drug release (∼40% in 2 h). However, no drug release from the Mef-Lipo was measured in the bile salt-free intestinal fluid or simulated saliva (0% in 3 h). These data suggest that drug release from the Mef-Lipo was mediated by a low pH and the presence of a surfactant. Pancreatic lipase did not degrade DSPC in the Mef-Lipo after 8 h of incubation nor induce Mef release from the liposomes, indicating that lipid digestion played a minor role for drug release from the Mef-Lipo. In order to improve long-term room temperature storage, the Mef-Lipo was lyophilized to obtain a solid formulation, which was completely dissolvable in water in 10 s and displayed similar in vitro profiles of release as the liquid form. The lyophilized Mef-Lipo was stable at room temperature for >3 months. In mice, orally delivered liquid and lyophilized Mef-Lipo displayed comparable absorption with bioavailability (BA) of 81-86%, while the absorption of the standard Mef suspension was significantly lower with BA of 70% and 20% decreased maximal plasma concentration and area under the curve. Our data suggest that the Mef-Lipo was a stable, palatable, and bioavailable formulation that might be suitable for pediatric use.

A Simple and Improved Active Loading Method to Efficiently Encapsulate Staurosporine into Lipid-Based Nanoparticles for Enhanced Therapy of Multidrug Resistant Cancer.

PURPOSE: This study was aimed at developing a new active loading method to stably encapsulate staurosporine (STS), a water insoluble drug, into lipid-based nanoparticles (LNPs) for drug targeting to tumors. METHODS: A limited amount of DMSO was included during the active loading process to prevent precipitation and facilitate the loading of insoluble STS into the aqueous core of a LNP. The drug loading kinetics under various conditions was studied and the STS-LNPs were characterized by size, drug-to-lipid ratio, drug release kinetics and in vitro potency. The antitumor efficacy of the STS-LNPs was compared with free STS in a mouse model. RESULTS: The drug loading efficiency reached 100% within 15 min of incubation at a drug-to-lipid ratio of 0.31 (mol) via an ammonium gradient. STS formed nano-aggregates inside the aqueous core of the LNPs and was stably retained upon storage and in the presence of serum. A 3-fold higher dose of the STS-LNPs could be tolerated by BALB/c mice compared with free STS, leading to nearly complete growth inhibition of a multidrug resistant breast tumor, while free STS only exhibited moderate activity. CONCLUSION: This simple and efficient drug loading method produced a stable LNP formulation for STS that was effective for cancer treatment.

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

Inhibition of the LSD1 (KDM1A) demethylase reactivates the all-trans-retinoic acid differentiation pathway in acute myeloid leukemia.

Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non-APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine-specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4(me2)) across the genome, but it did increase H3K4(me2) and expression of myeloid-differentiation-associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML.

Non-viral vector as vaccine carrier.

Over the last several years, advances in gene-based delivery technology arising from the field of gene therapy have helped revitalize the field of vaccine development. Genetic vaccination encoding antigen from bacteria, virus, and cancer has shown promise in protective humoral and cellular immunity; however, the potential disadvantages of naked DNA vaccine have reduced the value of the approach. To optimize antigen delivery efficiency as well as vaccine efficacy, the non-viral vector as vaccine carrier, for example, the cationic liposome, has shown particular benefits to circumvent the obstacles that both peptide/protein- and gene-based vaccines have encountered. Liposome-mediated vaccine delivery provides greater efficacy and safer vaccine formulation for the development of vaccine for human use. The success of the liposome-based vaccine has been demonstrated in clinical trials and further human trials are also in progress.