| 1. |
- Baudet, Aurélie, et al.
(author)
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Small Molecule Screening of Primary Human Acute Myeloid Leukemia Using Co-culture and Multiplexed FACS Analysis
- 2022
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In: Bio-protocol. - 2331-8325. ; 12:6
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Journal article (peer-reviewed)abstract
- Ex vivo culture of primary acute myeloid leukemia (AML) cells is notoriously difficult due to spontaneous differentiation and cell death, which hinders mechanistic and translational studies. To overcome this bottleneck, we have implemented a co-culture system, where the OP9-M2 stromal cells support the growth, but most notably limit the differentiation of primary AML cells, thus allowing for mechanistic studies in vitro. Additionally, the co-culture on OP9-M2 stromal is superior in preserving surface marker expression of primary (adult and pediatric) AML cells in comparison to stroma-free culture. Thus, by combining the co-culture with multicolor, high-throughput FACS, we can evaluate the effect of hundreds of small molecules on multi-parametric processes including: cell survival, stemness (leukemic stem cells), and myeloid differentiation on the primary AML cells at a single-cell level. This method streamlines the identification of potential therapeutic agents, but also facilitates combinatorial screening aiming, for instance, at dissecting the regulatory pathways in a patient-specific manner.
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| 2. |
- Hultmark, Simon, et al.
(author)
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Combinatorial molecule screening identifies a novel diterpene and the BET inhibitor CPI-203 as differentiation inducers of primary acute myeloid leukemia cells
- 2021
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In: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 106:10
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Journal article (peer-reviewed)abstract
- Combination treatment has proven effective for patients with acute promyelocytic leukemia, exemplifying the importance of therapy targeting multiple components of oncogenic regulation for a successful outcome. However, recent studies have shown that the mutational complexity of acute myeloid leukemia (AML) precludes the translation of molecular targeting into clinical success. Here as a complement to genetic profiling, we used unbiased, combinatorial in vitro drug screening to identify pathways that drive AML and to develop personalized combinatorial treatments. First, we screened 513 natural compounds on primary AML cells and identified a novel diterpene (H4) that preferentially induced differentiation of FLT3 wild-type AMLs, while FLT3-ITD/mutations conferred resistance. The responding samples to H4, displayed increased expression of myeloid markers, a clear decrease in the nuclear-cytoplasmic ratio and the potential of re-activation of the monocytic transcriptional program reducing leukemia propagation in vivo. By combinatorial screening using H4 and molecules with defined targets, we demonstrated that H4 induces differentiation by the activation of protein kinase C (PKC) signaling pathway, and in line with this, activates PKC phosphorylation and translocation of PKC to the cell membrane. Furthermore, the combinatorial screening identified a bromo- and extra-terminal domain (BET) inhibitor that could further improve H4-dependent leukemic differentiation in FLT3 wild-type monocytic AML. Taken together, this illustrates the value of an unbiased and multiplex screening platform for developing combinatorial therapeutic approaches for AML.
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| 3. |
- Hultmark, Simon
(author)
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Modeling normal and malignant hematopoiesis in vitro. To screen for extrinsic regulators and differentiation therapy.
- 2022
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Doctoral thesis (other academic/artistic)abstract
- The incredible thing with blood stem cells, also known as hematopoietic stem cells (HSC), is that they can restore normal hematopoiesis in patients that need a new blood system. Since a prerequisite for successful transplantation is immune compatibility, it requires large donor registries to find a suitable match for a recipient. Unfortunately, there is still a shortage of immune-compatible donors in these registries. Thus one potential approach to improve the quantity and quality of these registries is to expand HSC in umbilical cord blood units. However, robust in vitro expansion of human HSC is not possible yet. Thus, in vitro expansion of HSC is a high-value objective in hematological research.A common reason patients need a new blood system is blood cancer. An added benefit of transferring someone’s else blood system to a cancer patient is that the donor’s immune cells can help eradicate the cancer cells. Because the transfer of a new blood system is not without risks, as the donor’s immune cells also target normal tissues, physicians will only transplant when the cancer treatment is not potent enough to eradicate the cancer cells. Even though the cancer therapies of some AML subtypes are effective today, the prognosis of most cancer types would improve with new therapies. Thus, developing new therapies is another high-value objective in hematological research.Here we addressed both of these objectives by using a mesenchymal stroma-based co-culture model for culturing primary acute myeloid leukemia (AML) cells and HSC to identify differentiation therapy of AML (papers I and II), improve culture conditions of HSC (paper III), and for investigating synthetic lethality in AML (paper IV). Thus, the common thread of the four papers included in this thesis is the use of OP9M2 cells to model normal and malignant hematopoiesis.In paper I, we identified a natural product that induces differentiation in AML through activation of the PKC pathway. Moreover, we show that AML with FLT3-ITD or FLT3 mutations are resistant to differentiation, highlighting the importance of neutralizing the effect of mutated FLT3 in differentiation therapy. This study illustrates how small molecule screening and genetic profiling are powerful tools for developing personalized treatments.Paper II is a small molecule screening protocol based on the OP9M2 co-culture model of primary AML cells. With a flow-cytometry readout, the protocol is highly adjustable to different study objectives, including screening for novel therapeutic agents, drug repurposing, drug synergism, patient selection, mechanism of action analysis, and drug resistance. Methods such as these will continue to be crucial for developing new therapies to improve outcomes for many patient groups.In Paper III, we identified potential regulators of HSC, which we screened by shRNA knockdown in the OP9M2 model. However, it did not identify any candidates, likely due to a sub-optimal screening methodology. Still, the list of potential regulators could be helpful for similar studies. Improving in vitro culture conditions remains a high-value objective as cellular therapies will continue to be essential for treating hematological diseases.Paper IV shows that STAG1 and STAG2 have a synthetic lethal interaction in primary AML cells. Thus, targeting STAG1 or STAG2 in STAG1- or STAG2-null AML is potentially a new precision medicine for molecular targeted therapy. This study shows how an in-depth understanding of disease heterogeneity and subtype-specific weaknesses is critical for developing precision medicine.
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