Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - PNANOMED (Personalized Nanomedicines for Leukemia Patients)

Teaser

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder affecting myeloid hematopoiesis, caused by accumulation of genetic aberrations that result in increased self-renewal and proliferation, a block in differentiation, and reduced apoptosis. Although the majority of...

Summary

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder affecting myeloid hematopoiesis, caused by accumulation of genetic aberrations that result in increased self-renewal and proliferation, a block in differentiation, and reduced apoptosis. Although the majority of AML patients respond well to initial chemotherapy, the disease eventually relapses in the majority of patients, who then require treatments that eradicate the leukemia stem cell. Extensive efforts have been undertaken during the last 30 years to find better treatments. However, for the majority of AML patients the same chemotherapy as 30 years earlier is used, reflected by the dismal prognosis of this disease. To alleviate the burden this disease is putting on individuals, their families and society in general, we will decipher the interdependence of genetic mutations and develop nanoparticle/siRNA formulations to inhibit the network of genes that keeps the leukemic cell alive.

Our strategy is to apply cutting-edge technology to mouse models from primary human AML patients to identify the target proteins that sustain leukemia stem cell self-renewal and to develop effective nanomedicines against these targets. We strictly use primary AML models to study the functional hierarchy of genetic aberrations and to prioritize potential target molecules. A biobank of human transplantable AML xenografts is being established (WP1) and characterized by state-of-the-art genomic approaches (WP2). Genetic aberrations are being reversed by knockdown and, gene replacement strategies and functional consequences are being assessed in vivo (WP3). Most advanced nanoparticle/siRNA formulations and preparation tools have been employed to develop leukemia-specific nanomedicines (WP4 and 5). Thus, we will improve the understanding of AML biology to develop better treatment for our leukemia patients.

Work performed

We developed a biobank of patient-derived leukemia xenograft models that are serially transplantable and are genetically characterized. These models were used to evaluate the preclinical efficacy and pharmacokinetics of a novel small molecule inhibitor of mutant IDH1, which is found in six percent of AML patients and is currently evaluated in clinical trials. We also developed a new method to allow engraftment of patient derived cells, which previously did not engraft in mice.
We also developed the technology to genetically manipulate patient-derived cells in vivo. We use a genetic barcode to label lentiviral vectors, which carry different genes or shRNAs. We can then monitor the genetically modified cells by next generation sequencing and determine whether inhibition or normalization of a mutated gene has an anti-leukemic effect. These experiments are performed in vivo and therefore are as close as possible to the situation of a leukemia patient.
We established the safety and efficacy of lipid nanoparticle/siRNA formulations that were directed against human leukemia cells. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of leukemic xenograft models. By targeting BCR-ABL and TCF3-PBX1 fusion oncogenes we show a reduction of leukemic burden in our myeloid and lymphoid xenotransplant models, and demonstrate improved survival of acute lymphoblastic leukemia mice compared to control siRNA treated mice mediated by target knockdown. Our study provides proof-of-principle that fusion oncogene-specific RNAi therapeutics can be exploited to target leukemia cells and promise novel treatment options for leukemia patients.
The PNANOMED project is well underway and has contributed novel findings to leukemia research. We also collaborate with multiple other research groups and thereby disseminate our models, findings and technology.

Final results

Our biobank of patient-derived leukemia xenograft models is a unique source of highly relevant leukemias, which are carefully characterized for their genetic hierarchy in my laboratory. The relevance of these models is underscored by the multiple collaborations with other researchers, which build on these models.
In vivo screenings with shRNAs or cDNAs in patient-derived leukemia cells is hardly performed worldwide and thus our methodology represents a unique advance that will give us novel insights into the constitution of leukemia stem cells.
We generated proof of principle that siRNA mediated inhibition of leukemia oncogenes can delay a patient-derived leukemia in vivo. This takes previous studies one step further and suggests that our technology should be developed further for clinical use in leukemia patients.

During the next period of 2.5 years we expect critical insights into the relevance and function of the mutated genes found in AML patients. We will perform multiple shRNA- and cDNA-in vivo screenings and will identify the hierarchy and interdependence of the mutated genes in AML. We will also evaluate pharmacologic strategies to inhibit or restore the most relevant genes and will especially focus on combined targeting of multiple mutated genes.
We will develop additional siRNAs against AML oncogenes and will validate them in our xenograft models in vivo. We will also modify our lipid nanoparticles with leukemia specific ligands and evaluate whether this approach can improve the efficacy of siRNA delivery in leukemic cells in vivo.
In summary, the PNANOMED project has met important milestones and promises to make significant contributions beyond the state of the art for our leukemia patients.