Contributions
Abstract: S815
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 11:45 - 12:00
Location: Room A4
Background
Acute myeloid leukemia (AML) is characterized by an accumulation of immature myeloid blasts in the bone marrow. By providing cell-cell interactions and secreted factors, the bone marrow niche supports AML and normal hematopoietic stem and progenitor cells (HSPCs). A dysregulation of cytokines in the bone marrow microenvironment upon AML development contributes to the selective advantage of leukemia stem cells, a self-renewing population of leukemia cells that constitutes a chemo-resistant reservoir responsible for disease relapse.
Aims
To identify factors that regulate AML cells, we recently developed an in vitro cytokine screen using fluorescently labeled c-Kit+ leukemia cells mixed with corresponding normal bone marrow cells, allowing us to successfully identify both negative and positive regulators of AML cells (Peña-Martínez et al., Leukemia 2017). However, to assess effects on leukemia stem cells, there is a strong demand to improve such screens to evaluate the impact of cytokines on the leukemia-initiating capacity of cells more directly using an in vivo readout. A major challenge for combining ex vivo screens with in vivo read-out of stem cell function is the large number of experimental animals needed to provide meaningful data. Hence, new methods that allow for a multiplexed in vivo read-out of leukemia-initiating activity are needed.
Methods
To identify cytokines that regulate AML stem cells using a competitive in vivo read-out of leukemia-initiating activity, we generated lentiviral vectors harboring 11 genetic barcodes in an arrayed setting. This approach allows for labeling of leukemia cell populations with distinct molecular barcodes followed by exposure to separate experimental conditions. Each labeled cell population was stimulated with one cytokine ex vivo and after culture, leukemia cells from up to 11 cytokine conditions were pooled prior to in vivo competition. To trace the effects of the cytokines to the leukemia-initiating capacity of barcoded cells, the representation of individual barcodes in vivo was assessed using next-generation sequencing (NGS).
Results
With this approach, we assessed the effect of 114 murine cytokines on MLL-AF9 AML mouse cells and identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13) as a positive regulator of leukemia-initiating cells. By using a Tnfsf13-/- mouse, we confirmed that TNFSF13 supports leukemia initiation and also normal myelopoiesis by regulating granulocyte and macrophage progenitor (GMP) cell levels in the bone marrow. TNFSF13 was secreted by normal myeloid cells but not by AML mouse cells, suggesting that mature myeloid bone marrow cells support AML cells by secreting TNFSF13. TNFSF13 supported leukemia cell proliferation in an NF-κB-dependent manner and suppressed apoptosis. Moreover, TNFSF13 supported the growth and survival of several human myeloid leukemia cell lines, demonstrating that our findings translate to human disease.
Conclusion
Taken together, using arrayed molecular barcoding, we identified a previously unrecognized role of TNFSF13 as a positive regulator of primitive AML cells. The arrayed barcoded screening methodology is not limited to cytokines and leukemia, but can be extended to other types of ex vivo screens, where a multiplexed in vivo read-out of stem cell functionality is needed.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Leukemic Stem Cell, Screening
Abstract: S815
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 11:45 - 12:00
Location: Room A4
Background
Acute myeloid leukemia (AML) is characterized by an accumulation of immature myeloid blasts in the bone marrow. By providing cell-cell interactions and secreted factors, the bone marrow niche supports AML and normal hematopoietic stem and progenitor cells (HSPCs). A dysregulation of cytokines in the bone marrow microenvironment upon AML development contributes to the selective advantage of leukemia stem cells, a self-renewing population of leukemia cells that constitutes a chemo-resistant reservoir responsible for disease relapse.
Aims
To identify factors that regulate AML cells, we recently developed an in vitro cytokine screen using fluorescently labeled c-Kit+ leukemia cells mixed with corresponding normal bone marrow cells, allowing us to successfully identify both negative and positive regulators of AML cells (Peña-Martínez et al., Leukemia 2017). However, to assess effects on leukemia stem cells, there is a strong demand to improve such screens to evaluate the impact of cytokines on the leukemia-initiating capacity of cells more directly using an in vivo readout. A major challenge for combining ex vivo screens with in vivo read-out of stem cell function is the large number of experimental animals needed to provide meaningful data. Hence, new methods that allow for a multiplexed in vivo read-out of leukemia-initiating activity are needed.
Methods
To identify cytokines that regulate AML stem cells using a competitive in vivo read-out of leukemia-initiating activity, we generated lentiviral vectors harboring 11 genetic barcodes in an arrayed setting. This approach allows for labeling of leukemia cell populations with distinct molecular barcodes followed by exposure to separate experimental conditions. Each labeled cell population was stimulated with one cytokine ex vivo and after culture, leukemia cells from up to 11 cytokine conditions were pooled prior to in vivo competition. To trace the effects of the cytokines to the leukemia-initiating capacity of barcoded cells, the representation of individual barcodes in vivo was assessed using next-generation sequencing (NGS).
Results
With this approach, we assessed the effect of 114 murine cytokines on MLL-AF9 AML mouse cells and identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13) as a positive regulator of leukemia-initiating cells. By using a Tnfsf13-/- mouse, we confirmed that TNFSF13 supports leukemia initiation and also normal myelopoiesis by regulating granulocyte and macrophage progenitor (GMP) cell levels in the bone marrow. TNFSF13 was secreted by normal myeloid cells but not by AML mouse cells, suggesting that mature myeloid bone marrow cells support AML cells by secreting TNFSF13. TNFSF13 supported leukemia cell proliferation in an NF-κB-dependent manner and suppressed apoptosis. Moreover, TNFSF13 supported the growth and survival of several human myeloid leukemia cell lines, demonstrating that our findings translate to human disease.
Conclusion
Taken together, using arrayed molecular barcoding, we identified a previously unrecognized role of TNFSF13 as a positive regulator of primitive AML cells. The arrayed barcoded screening methodology is not limited to cytokines and leukemia, but can be extended to other types of ex vivo screens, where a multiplexed in vivo read-out of stem cell functionality is needed.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Leukemic Stem Cell, Screening