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Targeting PKC and BET induces differentiation of AML
above 60 years of age.2 The overall poor survival high- lights a dire need for better therapies.
During the past years, several targeted therapies have been developed, designed to uniquely target disease-spe- cific molecular events, such as mutant isocitrate dehy- drogenase proteins,3,4 BCR/ABL5 and PML/RARAα fusion proteins6 as well as FLT3-ITD mutations.7 Targeted ther- apies hold the promise of being superior to standard chemotherapy with increased specificity, improved effi- ciency, and reduced toxicity/side-effects.8 However, sin- gle-agent targeted therapies have had only moderate clinical success. For instance, targeting mutant IDH2 or FLT3-ITD AML with specific inhibitors produced initial molecular responses, but these promising early results were frequently followed by treatment resistance and relapse.3,9,10 Relapse and resistance could be explained by the expansion of malignant clones that were not depend- ent on the targeted mutation,11,12 indicating that single- agent targeted therapy would not be sufficient for leukemia clearance.
In line with this, combining arsenic salt with standard all-trans retinoic acid therapy for acute promyelocytic leukemia enabled simultaneous activation of both RARα-dependent granulocytic differentiation and the PML-dependent apoptosis/senescence pathways.13-15 This simultaneous targeting of multiple drivers is now the standard treatment for patients carrying PML-RARα rearrangements and results in disease clearance in more than 90% of these patients who previously had poor prognoses.6 This demonstrated the need for tailored treatment targeting multiple mechanisms driving the dis- ease. However, next-generation sequencing has shown that most cases of AML have a far more complex muta- tional landscape with an average of 13 mutations per sample (excluding alterations in noncoding regions)16 as well as complex karyotypes, highlighting the challenge of translating multiple-targeted therapy into clinical suc- cess for the treatment of AML.
As a complement to genetic profiling, unbiased in vitro drug screening can be used to reveal pathways that drive disease and identify novel therapeutic targets. By com- bining drug screening and the multiplex power of flow cytometry, we were also previously able to simultane- ously identify compounds with cytotoxicity and differ- entiation-inducing potential, defining a paradigm for high throughput delineation of potential combination therapies.17 Furthermore, unlike xenograft models, a short culture (<1 week) can efficiently maintain the poly- clonality of AML, making in vitro models and high throughput drug screening a powerful tool for develop- ing personalized therapies for AML.18,19
Here, to identify patient-specific combinatorial treat- ment options, we improved our stroma co-culture model for mechanistic and combinatorial screening of primary AML cells.17 We identified H4, a novel, natural com- pound that induced differentiation of FLT3 wild-type pri- mary AML samples, while FLT3-ITD/mutated AML were found to be resistant to H4 treatment. Using com- binatorial screening, we established that H4 induced dif- ferentiation by activation of protein kinase C (PKC) sig- naling and that, for FLT3 wild-type monocytic AML sam- ples,theeffectofH4wasfurtherenhancedbyabromo- and extra-terminal domain (BET) inhibitor, demonstrat- ing the potential of unbiased approaches in the develop- ment of personalized treatments.
Methods
The methods are described in detail in the Online Supplementary Material.
Collection of healthy bone marrow, umbilical cord blood, and acute myeloid leukemia samples
Samples were collected in Swedish hospitals, in accordance with the Declaration of Helsinki and with the approval of the local ethi- cal committees (dnr 2014/596, 826/2004 and 2010/1893-31/2). Mononuclear cells from umbilical cord blood and healthy bone marrow were enriched for CD34+ cells and primary AML samples for mononuclear cells before freezing. The characteristics of the patients’ samples are presented in Table 1 and Online Supplementary Table S1.
Small molecule libraries
The natural product library was obtained from AnalytiCon Discovery (Potsdam, Germany), as a 10 mM stock solution in dimethylsulfoxide (DMSO). H4 product #NP-000694. The anti- cancer drug library was obtained from Selleckchem (L3000).
Small molecule screening
Small molecule screening has been previously described.17 Briefly, primary hematopoietic cells were plated on irradiated OP9M2 stro- mal cells in medium supplemented with human cytokines (stem cell factor, thrombopoietin, FLT3, interleukin 6, and interleukin 3). After 36 to 48 h, compounds were added at the final concentration of 0.5 mM and 10 mM. AML cell lines were treated for 3 days and primary AML samples for 4 days before analysis.
Flow cytometry analysis
Cultured cells were transferred to 96-well round-bottomed plates, washed, and then stained with anti-human monoclonal anti- bodies (CD11b, CD15, CD64). Cells were analyzed using a FACSCanto II analyzer with a high-throughput unit (Becton Dickinson).
Long-term culture
Every 4 days, the medium was changed completely by adding new medium containing H4 10 mM or DMSO. Cells were immunophenotyped and a volumetric cell count performed using flow cytometry.
Cell cycle inhibition
AML-3 cells were pre-treated for 5 days with palbociclib 5 mM or DMSO before H4 was added with a complete change of medium. On days 5 and 9, cells were anlyzed by immunostaining, volumet- ric cell count, and cell cycle status using DAPI and PE Mouse Anti- Ki-67 Set (BioLegend).
In vitro treatment, transplantation into NRGS mice and analysis of engraftment
Cultured cells from an equal number of wells per treatment group were transplanted into pre-conditioned (600 cGy radiation) NRGS mice. When mice from the DMSO group showed signs of sickness, mice from both groups were sacrificed. Spleen and bone morrow were collected, stained for human CD45 and CD33 (BioLegend) and analyzed by flow cytometry.
Protein kinase C translocation assay
HEK-293 cells were transfected with plasmids encoding EGFP- tagged full-length PKC20 and treated with 40 mM of H4. Translocation of PKC-EGFP from the cytoplasm to the plasma membrane was measured using live imaging on a Zeiss 780 confo- cal laser scanning microscope.
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