ALBERT

Description: Background: Despite the increase in patients' survival over the last years, multiple myeloma (MM) remains incurable, being persistence of cancer stem cells (CSCs) a probable cause of drug resistance and disease relapse. It is possible to isolate these cells using surface antigen expression pattern (CD19+/CD34+/CD138-) and the activity of an enzyme from aldehyde dehydrogenase (ALDH) family (Boucher et al., 2012). We believe that using CD19 as potential marker of MM-CSCs makes CAR-T cell therapy against CD19 an option to eradicate residual MM disease. Aims: To isolate and characterize immunophenotypically, functionally and by gene expression the MM-CSCs derived from bone marrow (BM) samples of newly-diagnosed MM patients, focusing on identification of possible therapeutic targets. Methods: BM aspirates were collected and CD138+ cells were separated by magnetic sorting. The remaining cells were submitted to sorting by flow cytometry on FACSAria II (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), labeled with anti-CD19 Pacific Blue (Invitrogen, Carlsbad, CA, USA), anti-CD34 PE Cy7 and anti-CD138 APC (both from Becton, Dickinson and Company, Franklin Lakes, NJ, USA) antibodies, in addition to Aldefluor™ reagent (StemCell Technology, Vancouver, British Columbia, Canada). RNA was extracted and pre-amplified for PCR array analysis using the RT² Profiler™ PCR Array Human Cancer Stem Cells(Qiagen, Hilden, Germany) to assess the expression profile of 84 genes related to cancer stem cells, and the results were evaluated with the online software provided by the platform manufacturer. Results: MM-CSCs (CD34+/CD19+/CD138-/ALDH1+) were isolated by flow cytometry from MM samples and presented median of 1,748.5 events (ranging from 56 to 16,633, n = 16). For comparison purposes, CD138+ MM tumor cells were isolated and used as "control group" (median of events 72,904, ranging from 1,536 to 312,504, n = 15). RNA from 16 MM-CSC samples and 6 controls were analyzed by qPCR. Considering 2-ΔCt calculation (GAPDH as normalizer) and fold change of 2, 11 genes were considered differentially expressed in MM-CSCs when compared to tumor plasma cells (p

Description: Background: Despite the absence of JAK1 and JAK2 mutations in multiple myeloma (MM), high levels of IL-6 constitutively activate the JAK/STAT pathway promoting survival and proliferation of tumor cells. Therefore, pharmacological inhibition of JAK proteins can be a potentially therapeutic strategy for myeloma treatment. Aims: 1) to identify expression of JAK1 and JAK2 in MM cell lines and in recently diagnosed MM patients; 2) to perform functional in vitro studies in MM cell lines treated with JAK/STAT pathway inhibitor (ruxolitinib), associated with drugs currently used in MM first line treatment (bortezomib, lenalidomide and dexamethasone), with and without co-culture with normal stromal cells; 3) to evaluate global gene expression of JAK/STAT pathway in cell lines treated with ruxolitinib to elucidate its mechanism of action in MM. Methods: JAK1 and JAK2 expression were analyzed in four cell lines (RPMI-8226, U266, SKO-007 and SKM-M2) and in bone marrow samples from 30 MM patients and 3 healthy controls by real time PCR. After IC50 calculation, drugs concentrations were: bortezomib (B) 10 nM for both RPMI-8226 and U266 cell lines; ruxolitinib (R) 30 µM for RPMI-8226 and 40 µM for U266 cell lines; lenalidomide (L) 10 µM for both cell lines; and dexamethasone (D) 1 µM for both cell lines. Apoptosis and cell cycle were evaluated by flow cytometry. PCR array for 92 JAK/STAT pathway related genes (Taqman® Array Human JAK/STAT Pathway, Applied Biosystems, Foster City, CA, USA) was performed in RPMI-8226 and U266 wild type and B+R treated cell lines, in duplicates. Results: Among the four cell lines, U266 presented the highest expression of JAK1 and JAK2 genes. JAK1 was overexpressed in 27% and JAK2 in 57% of 30 MM patients (considering at least 2-fold increase). After B+R treatment, RPMI-8226 showed increased number of cells in SubG0 phase (p