Efficient Chimeric Antigen Receptor T-Cell Generation Starting with Leukoreduction System Chambers of Thrombocyte Apheresis Sets.

Introduction: Primary human blood cells represent an essential model system to study physiology and disease. However, human blood is a limited resource. During healthy donor plateletpheresis, the leukoreduction system chamber (LRSC) reduces the leukocyte amount within the subsequent platelet concentrate through saturated, fluidized, particle bed filtration technology. Normally, the LRSC is discarded after apheresis is completed. Compared to peripheral blood, LRSC yields 10-fold mononuclear cell concentration. Methods: To explore if those retained leukocytes are attractive for research purposes, we isolated CD3+ T cells from the usually discarded LRSCs via density gradient centrifugation in order to manufacture CD19-targeted chimeric antigen receptor (CAR) T cells. Results: Immunophenotypic characterization revealed viable and normal CD4+ and CD8+ T-cell populations within LRSC, with low CD19+ B cell counts. Magnetic-activated cell sorting (MACS) purified CD3+ T cells were transduced with CD19 CAR-encoding lentiviral self-inactivating vectors using concentrated viral supernatants. Robust CD19 CAR cell surface expression on transduced T cells was confirmed by flow cytometry. CD19 CAR T cells were further enriched through anti-CAR MACS, yielding 80% CAR+ T-cell populations. In vitro CAR T cell expansion to clinically relevant numbers was achieved. To prove functionality, CAR T cells were co-incubated with the human CD19+ B cell precursor leukemia cell line Nalm6. Compared to unmodified T cells, CD19 CAR T cells effectively eradicated Nalm6 cells. Conclusion: Taken together, we can show that lymphocytes isolated from LRSCs of plateletpheresis sets can be efficiently used for the generation of functional CAR T cells for experimental purposes.

Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo.

BACKGROUND: Acute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells. METHODS: To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR‒Cas9 pipeline in PDX models in vivo. RESULTS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo. CONCLUSIONS: These findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias.

Defined Human Leukemic CD34+ Liquid Cultures to Study HDAC/Transcriptional Repressor Complexes.

Defined human primary cell model systems with growth dependence on oncogenes are highly requested to investigate tumor pathogenesis and to validate pharmacological inhibitors that specifically target oncoproteins and their executing protein complex partners. In acute myeloid leukemia (AML), transcription factors such as RUNX1 and MLL1, which are important for normal blood cell development, frequently harbor mutations including chromosomal translocations with other coding genes, resulting in tumor-promoting gain-of-function fusion proteins. These oncoproteins completely modify transcriptional programs, thereby inducing malignant cell phenotypes. A common theme of the chimeric gene products is their physical interaction with a variety of chromatin-modifying effector molecules, including histone acetyltransferases (HATs) and histone deacetylases (HDACs). These aberrant multiprotein machineries disturb gene expression and promote malignant cell growth. In this chapter, we briefly summarize the current understanding regarding AML-associated oncogene-driven human CD34+ blood progenitor cell expansion in ex vivo liquid cultures. We provide a step-by-step protocol to establish oncogene-induced human CD34+ blood progenitor cell cultures suitable to analyze the impact of transcriptional repressor/HDAC activity in these human AML cell models.

Deciphering Acute Myeloid Leukemia Associated Transcription Factors in Human Primary CD34+ Hematopoietic Stem/Progenitor Cells.

Hemato-oncological diseases account for nearly 10% of all malignancies and can be classified into leukemia, lymphoma, myeloproliferative diseases, and myelodysplastic syndromes. The causes and prognosis of these disease entities are highly variable. Most entities are not permanently controllable and ultimately lead to the patient's death. At the molecular level, recurrent mutations including chromosomal translocations initiate the transformation from normal stem-/progenitor cells into malignant blasts finally floating the patient's bone marrow and blood system. In acute myeloid leukemia (AML), the so-called master transcription factors such as RUNX1, KMT2A, and HOX are frequently disrupted by chromosomal translocations, resulting in neomorphic oncogenic fusion genes. Triggering ex vivo expansion of primary human CD34+ stem/progenitor cells represents a distinct characteristic of such chimeric AML transcription factors. Regarding oncogenic mechanisms of AML, most studies focus on murine models. However, due to biological differences between mice and humans, findings are only partly transferable. This review focuses on the genetic manipulation of human CD34+ primary hematopoietic stem/progenitor cells derived from healthy donors to model acute myeloid leukemia cell growth. Analysis of defined single- or multi-hit human cellular AML models will elucidate molecular mechanisms of the development, maintenance, and potential molecular intervention strategies to counteract malignant human AML blast cell growth.