Evaluation of phenotypic heterogeneity and the role of MRC2 in leukemic stem cells

Acute myeloid leukemia (AML) is a blood malignancy affecting people from all age categories yet most frequently elderly people (older than 65-year-old). Therapy options have improved over the last decades but AML still remain lethal in many cases despite many patients achieving apparent remission. Indeed, patients often relapse and such reoccurrence becomes harder to treat due to the development of therapy resistance and the selection of specific more aggressive subclones. AML was the first malignancy to support the theory of cancer stem cells and is widely described for its hierarchical structure. In fact, AML is known to originate and relapse from a rare cell subpopulation of leukemic cells called leukemic stem cells (LSC). AML and their LSC are very heterogeneous on the transcriptomic and phenotypic level which makes it challenging to eradicate these cells. Currently, efforts are made by many research groups to determine how to selectively and specifically target LSC. Not only would this improve our knowledge on their biology but this could potentially greatly improve clinical care. In this thesis, we improved knowledge on LSC biology and highlighted a potential new vulnerability. We discovered that MRC2 is overexpressed in AML and specifically enriched cells with LSC properties. RNA-sequencing of MRC2+ vs. MRC2- cells revealed the former population to have, on the transcriptomic level, a stemness signature that is highly concordant with our in vitro and in vivo assays indicating positive cells to be more clonogenic in colony forming unit (CFU) assay and patient-derived xenograft (PDX) models, respectively. MRC2+ cells are functionally involved in collagen uptake, which engages cells metabolically. In depth in silico analyses revealed proline dehydrogenase (PRODH) as overexpressed in MRC2+ LSC. Importantly, PRODH inhibition impaired cell viability and stemness (with no effect on healthy cells) and synergized with the BCL-2 inhibitor venetoclax. Furthermore, TGF-β1 was discovered as a strong inducer of MRC2 expression/function in AML. In a related second project, variables influencing leukemic engraftment in PDX models were studied in an attempt to further improve these models that are essential for functional studies of human AML. Noticeably, we discovered an improved leukemic engraftment in female vs. male NSG animals when injected with the exact same cell dose of primary sample in both primary and secondary transplant assays. This importantly made us reconsider how to continue with PDX models and, by only relying on female NSG mice, we further discovered that AML samples from the adverse risk group, with high FLT3-ITD ratio or with high LSC content had shorter time to engraft in female NSG mice. Our extended incubation period allowed us to appreciate engraftment in samples previously considered non-engraftable such as from favorable molecular risk groups. These observations are helpful as they mimic well clinical settings and furthermore allow comprehensive studies of AML biology in in vivo settings

Time Series of Bio-Optical Properties in a Subtropical Gyre: Implications for the Evaluation of Interannual Trends of Biogeochemical Properties

With a validated Quasi‐Analytical Algorithm, an 11 year (1998–2008) monthly time series of the primary optical properties of waters in the center of the South Pacific gyre was developed from Sea‐viewing Wide Field‐of‐view Sensor (SeaWiFS) and Moderate Resolution Imaging Spectroradiometer (MODIS). Also derived are chlorophyll a (Chl a) concentrations with the operational empirical algorithms for SeaWiFS and MODIS. The optical properties include the absorption coefficient (at 443 nm) of phytoplankton (aph) and that of the combination of detritus and gelbstoff (adg). From these time series, we further derived their annual background (summer low) and seasonal intensity (the difference between winter high and summer low). These time series show that (1) the optical properties have different seasonal and interannual variations, indicating different dynamics of these properties in the subtropical gyre; (2) there is a decreasing trend (r2 = 0.24) of the background aph in the 1998–2008 period and an increasing trend of the aph seasonal intensity (r2 = 0.11) for this period, and both trends are not statistically significant; (3) the aph time series agrees with the Chl a time series at the seasonal scale, but differs with respect to interannual variations; and (4) different interannual trends could be inferred with different time frames. These results emphasize that it is difficult to draw unequivocal conclusions about long‐term trends of biogeochemical properties in the oceans with the current relatively short bio‐optical records. To clarify and predict such trends, it is critical to get a full account of the forces that are responsible for the seasonal and interannual variations of these properties

Time Series of Bio-Optical Properties in a Subtropical Gyre: Implications for the Evaluation of Interannual Trends of Biogeochemical Properties

With a validated Quasi‐Analytical Algorithm, an 11 year (1998–2008) monthly time series of the primary optical properties of waters in the center of the South Pacific gyre was developed from Sea‐viewing Wide Field‐of‐view Sensor (SeaWiFS) and Moderate Resolution Imaging Spectroradiometer (MODIS). Also derived are chlorophyll a (Chl a) concentrations with the operational empirical algorithms for SeaWiFS and MODIS. The optical properties include the absorption coefficient (at 443 nm) of phytoplankton (aph) and that of the combination of detritus and gelbstoff (adg). From these time series, we further derived their annual background (summer low) and seasonal intensity (the difference between winter high and summer low). These time series show that (1) the optical properties have different seasonal and interannual variations, indicating different dynamics of these properties in the subtropical gyre; (2) there is a decreasing trend (r2 = 0.24) of the background aph in the 1998–2008 period and an increasing trend of the aph seasonal intensity (r2 = 0.11) for this period, and both trends are not statistically significant; (3) the aph time series agrees with the Chl a time series at the seasonal scale, but differs with respect to interannual variations; and (4) different interannual trends could be inferred with different time frames. These results emphasize that it is difficult to draw unequivocal conclusions about long‐term trends of biogeochemical properties in the oceans with the current relatively short bio‐optical records. To clarify and predict such trends, it is critical to get a full account of the forces that are responsible for the seasonal and interannual variations of these properties

Penetration of UV-visible Solar Radiation in the Global Oceans: Insights from Ocean Color Remote Sensing

Penetration of solar radiation in the ocean is determined by the attenuation coefficient (Kd(λ)). Following radiative transfer theory, Kd is a function of angular distribution of incident light and water\u27s absorption and backscattering coefficients. Because these optical products are now generated routinely from satellite measurements, it is logical to evolve the empirical Kd to a semianalytical Kd that is not only spectrally flexible, but also the sun-angle effect is accounted for explicitly. Here, the semianalytical model developed in Lee et al. (2005b) is revised to account for the shift of phase function between molecular and particulate scattering from the short to long wavelengths. Further, using field data collected independently from oligotrophic ocean to coastal waters covering \u3e99% of the Kd range for the global oceans, the semianalytically derived Kd was evaluated and found to agree with measured data within ∼7–26%. The updated processing system was applied to MODIS measurements to reveal the penetration of UVA-visible radiation in the global oceans, where an empirical procedure to correct Raman effect was also included. The results indicated that the penetration of the blue-green radiation for most oceanic waters is ∼30–40% deeper than the commonly used euphotic zone depth; and confirmed that at a depth of 50–70 m there is still ∼10% of the surface UVA radiation (at 360 nm) in most oligotrophic waters. The results suggest a necessity to modify or expand the light attenuation product from satellite ocean-color measurements in order to be more applicable for studies of ocean physics and biogeochemistry

Penetration of UV-Visible Solar Radiation in the Global Oceans: Insights from Ocean Color Remote Sensing

Penetration of solar radiation in the ocean is determined by the attenuation coefficient (K-d()). Following radiative transfer theory, K-d is a function of angular distribution of incident light and water\u27s absorption and backscattering coefficients. Because these optical products are now generated routinely from satellite measurements, it is logical to evolve the empirical K-d to a semianalytical K-d that is not only spectrally flexible, but also the sun-angle effect is accounted for explicitly. Here, the semianalytical model developed in Lee et al. (2005b) is revised to account for the shift of phase function between molecular and particulate scattering from the short to long wavelengths. Further, using field data collected independently from oligotrophic ocean to coastal waters covering \u3e99% of the K-d range for the global oceans, the semianalytically derived K-d was evaluated and found to agree with measured data within approximate to 7-26%. The updated processing system was applied to MODIS measurements to reveal the penetration of UVA-visible radiation in the global oceans, where an empirical procedure to correct Raman effect was also included. The results indicated that the penetration of the blue-green radiation for most oceanic waters is approximate to 30-40% deeper than the commonly used euphotic zone depth; and confirmed that at a depth of 50-70 m there is still approximate to 10% of the surface UVA radiation (at 360 nm) in most oligotrophic waters. The results suggest a necessity to modify or expand the light attenuation product from satellite ocean-color measurements in order to be more applicable for studies of ocean physics and biogeochemistry

Time Series of Bio-Optical Properties in a Subtropical Gyre: Implications for the Evaluation of Interannual Trends of Biogeochemical Properties

With a validated Quasi‐Analytical Algorithm, an 11 year (1998–2008) monthly time series of the primary optical properties of waters in the center of the South Pacific gyre was developed from Sea‐viewing Wide Field‐of‐view Sensor (SeaWiFS) and Moderate Resolution Imaging Spectroradiometer (MODIS). Also derived are chlorophyll a (Chl a) concentrations with the operational empirical algorithms for SeaWiFS and MODIS. The optical properties include the absorption coefficient (at 443 nm) of phytoplankton (aph) and that of the combination of detritus and gelbstoff (adg). From these time series, we further derived their annual background (summer low) and seasonal intensity (the difference between winter high and summer low). These time series show that (1) the optical properties have different seasonal and interannual variations, indicating different dynamics of these properties in the subtropical gyre; (2) there is a decreasing trend (r2 = 0.24) of the background aph in the 1998–2008 period and an increasing trend of the aph seasonal intensity (r2 = 0.11) for this period, and both trends are not statistically significant; (3) the aph time series agrees with the Chl a time series at the seasonal scale, but differs with respect to interannual variations; and (4) different interannual trends could be inferred with different time frames. These results emphasize that it is difficult to draw unequivocal conclusions about long‐term trends of biogeochemical properties in the oceans with the current relatively short bio‐optical records. To clarify and predict such trends, it is critical to get a full account of the forces that are responsible for the seasonal and interannual variations of these properties

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC