Understanding mechanisms of action of CPX-351 (Vyxeos) in acute myeloid leukaemia (AML): Focus on the leukaemic stem cell

Supervisors: Mhairi Copland, Helen Wheadon, Heather Jorgensen (University of Glasgow)

Collaborator: Sylvie Freeman (University of Birmingham)

Background
Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder which arises following progenitor cell acquisition of abnormal self-renewal potential giving rise to a leukaemic stem cell (LSC) (1). It is the most common malignant myeloid disorder in adults, with an annual incidence of approximately 3.8 per 100,000. Untreated AML typically results in bone marrow failure, leading to fatal infection, bleeding, or organ infiltration, within 1 year of diagnosis, but often within weeks to months. Treatment and prognosis vary significantly between the subtypes, overall survival varying from 20-47%, depending on subtype, mutational status and age (2). Treatment is associated with considerable morbidity and mortality however, and a cure for the majority of adults remains elusive. Additional/ alternative therapies are therefore a focus of intense research.

CPX-351 (Vyxeos) is a liposomal formulation of cytarabine and daunorubicin, packaged at a fixed 5:1 molar ratio within 100nm diameter liposomes, designed to deliver synergistic drug ratios to leukaemia cells (3, 4). Delivery of a maximally synergistic drug ratio enhances treatment efficacy, and preclinical studies have reported improved outcomes in animal leukaemia models with CPX-351 compared to cytarabine and daunorubicin administered conventionally (5). Further in vitro studies have demonstrated the preferential intracellular accumulation and cytotoxicity of CPX-351 for AML stem/progenitor cells compared to normal stem/progenitor cells (6). Importantly, in these in vitro studies, normal haemopoietic stem/progenitor cells were 5-fold less sensitive to CPX-351 than conventional cytarabine and daunorubicin, providing an important therapeutic window. The mechanism(s) behind the selective toxicity of CPX-351 for AML stem/progenitor cells is unclear and warrants further investigation. One avenue worth exploring is the altered metabolism reported in AML LSC compared to HSC and whether this results in higher up-take of liposome encapsulated drugs.
Clinical trials have demonstrated efficacy of CPX-351 in both newly diagnosed and relapsed/ refractory AML. In the front-line setting in elderly AML patients, CPX-351 produced higher response rates (66.7% versus 51.2%) than conventional cytarabine/daunorubicin combination, particularly among patients with secondary AML (7). In the relapse setting, CPX-351 demonstrated a trend towards improvement in efficacy outcomes as compared with a conventional salvage regimen of cytarabine/anthracycline (8, 9). Based on these clinical trial data, CPX-351 has now received FDA/EMEA approval for the treatment of patients with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes.

Aims
1. Elucidate the effect of CPX-351 (Vyxeos) on the functional properties of different leukaemic stem and progenitor cell populations in AML. Monitor metabolic rates of AML Stem/Progenitor cells and up-take of fluorescently labelled CPX-351 in the different cell populations.
2. Define the molecular changes associated with CPX-351 treatment in AML stem/progenitor cells. Are the molecular changes seen with CPX-351 different to those seen with conventional cytarabine and daunorubicin?
3. Explore the effect of CPX-351 on LSC niche cells and interactions between AML LSCs and stromal cells.
4. Based on results from aims 2 & 3, validate the identification of novel targets in samples from patients treated with CPX-351 on the NCRI AML19 clinical trial, and determine if these targets may be exploited in combination with CPX-351, leading to synergistic actions against AML LSCs.

Plan of investigation
Cells:
Under existing COREC approval, our research laboratory has established a tissue bank consisting of normal (control; obtained by leukapheresis), molecularly typed primary AML and bone marrow stromal cell samples. In addition, several AML cell lines are available in-house.
Aims 1 & 2:
Primary AML and normal haemopoietic cells will be sorted into different stem and progenitor cell populations using multiparameter FACS. The isolated populations will be treated with CPX-351 or conventional cytarabine/daunorubicin at an equivalent 5:1 molar ratio. Trypan blue cell counts, apoptosis assays, cell cycle assessment and colony forming cell assays will be performed to understand the short term effects of either CPX-351 or conventional cytarabine/daunorubicin on primitive AML cells. To assess the effect of CPX-351 on self-renewal of LSC, LTC-IC assays and re-plating efficiency of CFC assays will be performed. Seahorse technology will be used to measure the metabolism of the different cell populations and fluorescently labelled CPX-351 will be monitored by Flow-Cytometry.
To define the molecular changes associated with treatment with CPX-351, we will perform single cell RNAseq using the Smartseq2 protocol (10) on individual cells from the different stem and progenitor cell populations +/- CPX-351 or conventional cytarabine/daunorubicin. Raw reads will be subject to quality assessment (e.g., fastQC/multiQC) with subsequent trimming (e.g., TrimGalore) if necessary; alignment and count matrices will be generated (e.g., kallisto); and differential expression calculated (e.g., Sleuth). This initial in silico work will allow us to identify molecular changes associated with treatment response, characterise quiescent LSC persisting during treatment, and evaluate any differences between the molecular changes associated with CPX-351 or conventional cytarabine/daunorubicin to explain the differences in effect on normal stem/progenitor cells. We will further exploit the unique value of scRNA-seq data by identifying and molecularly characterising subpopulations of cells shown to emerge following treatment (via dimensionality reduction and clustering algorithms) and explore variability in gene- (e.g., MAST) and pathway-level (e.g., PAGODA) to assess to what extent transcriptional heterogeneity might define response to treatment.
Aim 3:
In co-culture experiments, using stromal cell lines and mesenchymal stem cells, we will study the effects of CPX-351 on bone marrow stromal cells, and any alterations with the interactions between stromal cells and LSC. Here, we will also utilise a novel bone marrow-like niche model, which comprises of nanomagnetically elevated mesenchymal stem cells cultured as multicellular spheroids within a type 1 collagen gel (11).
Aim 4:
In collaboration with Professor Sylvie Freeman, University of Birmingham, and in agreement with the Sponsor, we will data mine results from the AML19 trial to confirm molecular features associated with response or lack of response to CPX-351. Immunophenotypic stem/progenitor cell profiling incorporated into standard measurable residual disease monitoring will characterise in real-time resistant cell subpopulations following patient treatment. There is the possibility to incorporate CyTOF deep phenotyping and single cell RNAseq of selected diagnostic and post CPX-351 patient samples to further validate and extend in vitro data. Using novel small molecules or re-purposed drugs, we will test these targets in AML samples in vitro (cell counts, apoptosis, cell cycle, CFC and LTC-IC assays), to identify the most promising combinations with CPX-351 for in vivo testing.

Training Outcomes
The project will provide exposure to a broad array of cellular and molecular techniques including: processing and culturing cell lines and primary cells from patients; in vitro co-culture systems; flow cytometry; Western blotting; RNA extraction and RT-PCR techniques; Q-PCR and mini-array Taqman-based PCR, including single cell PCR; RNAseq sample preparation and data analysis; cellular drug treatment; cell cycle, proliferation and apoptosis assays; and NSG xenograft assays. In addition, the student will be fully trained in dealing with the safety and ethical issues of working with human samples. Thus the student will become highly skilled in a range of in vitro techniques and in vivo models in the pre-clinical development of novel cancer therapies.

References
1. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature medicine. 1997 Jul;3(7):730-7. PubMed PMID: 9212098.
2. Estey E, Dohner H. Acute myeloid leukaemia. Lancet. 2006 Nov 25;368(9550):1894-907. PubMed PMID: 17126723.
3. Mayer LD, Harasym TO, Tardi PG, Harasym NL, Shew CR, Johnstone SA, et al. Ratiometric dosing of anticancer drug combinations: controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice. Molecular cancer therapeutics. 2006 Jul;5(7):1854-63. PubMed PMID: 16891472.
4. Tardi P, Johnstone S, Harasym N, Xie S, Harasym T, Zisman N, et al. In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy. Leukemia research. 2009 Jan;33(1):129-39. PubMed PMID: 18676016.
5. Lim WS, Tardi PG, Dos Santos N, Xie X, Fan M, Liboiron BD, et al. Leukemia-selective uptake and cytotoxicity of CPX-351, a synergistic fixed-ratio cytarabine:daunorubicin formulation, in bone marrow xenografts. Leukemia research. 2010 Sep;34(9):1214-23. PubMed PMID: 20138667.
6. Kim HP, Gerhard B, Harasym TO, Mayer LD, Hogge DE. Liposomal encapsulation of a synergistic molar ratio of cytarabine and daunorubicin enhances selective toxicity for acute myeloid leukemia progenitors as compared to analogous normal hematopoietic cells. Experimental hematology. 2011 Jul;39(7):741-50. PubMed PMID: 21530609.
7. Lancet JE, Cortes JE, Hogge DE, Tallman MS, Kovacsovics TJ, Damon LE, et al. Phase 2 trial of CPX-351, a fixed 5:1 molar ratio of cytarabine/daunorubicin, vs cytarabine/daunorubicin in older adults with untreated AML. Blood. 2014 May 22;123(21):3239-46. PubMed PMID: 24687088. Pubmed Central PMCID: 4624448.
8. Cortes JE, Goldberg SL, Feldman EJ, Rizzeri DA, Hogge DE, Larson M, et al. Phase II, multicenter, randomized trial of CPX-351 (cytarabine:daunorubicin) liposome injection versus intensive salvage therapy in adults with first relapse AML. Cancer. 2015 Jan 15;121(2):234-42. PubMed PMID: 25223583. Pubmed Central PMCID: 5542857.
9. Feldman EJ, Lancet JE, Kolitz JE, Ritchie EK, Roboz GJ, List AF, et al. First-in-man study of CPX-351: a liposomal carrier containing cytarabine and daunorubicin in a fixed 5:1 molar ratio for the treatment of relapsed and refractory acute myeloid leukemia. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2011 Mar 10;29(8):979-85. PubMed PMID: 21282541. Pubmed Central PMCID: 4520927.
10. Giustacchini A, Thongjuea S, Barkas N, Woll PS, Povinelli BJ, Booth CAG, et al. Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia. Nature medicine. 2017 Jun;23(6):692-702. PubMed PMID: 28504724.
11. Lewis EE, Wheadon H, Lewis N, Yang J, Mullin M, Hursthouse A, et al. A Quiescent, Regeneration-Responsive Tissue Engineered Mesenchymal Stem Cell Bone Marrow Niche Model via Magnetic Levitation. ACS nano. 2016 Sep 27;10(9):8346-54. PubMed PMID: 27602872.

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