After 48 h (day 12), the live ALL cells were counted using trypan blue dye exclusion method and the % viability was calculated. 2.8. Taken together, our study demonstrates the utility of concomitantly targeting different critical regulatory pathways to induce cell death in drug resistant ALL cells. co-culture model of ALL cells with either primary human-derived BM stromal cells or osteoblasts (components of the BM niche) [9]. From this co-culture we characterized a drug resistant sub-population of leukemic cells referred to as phase dim (PD), based on their lack of light refraction coincident with their migration beneath adherent layers of stroma or osteoblasts. The PD tumor cells are used to model cells that contribute to MRD based on phenotypic similarities [9]. Using this niche-based Foliglurax monohydrochloride co-culture model, we have reported that primary ALL samples, or ALL cells in co-culture with the BM cellular components, have reduced BCL6 expression in the PD cell population [10]. Furthermore, reduction in BCL6 resulted in disruption of cell cycle progression, with cyclin D3-dependent accumulation of cells in the G0/G1 phase. The importance of BCL6 in maintaining cell quiescence, drug resistance and the resulting MDR phenotype was further validated by demonstrating significant event free survival in mice treated with a combination of caffeine (stabilizer of BCL6) and cyatarabine (Ara-C) when compared to mice treated with Ara-C alone [10]. BCL6 has also been shown to be a master regulator of glycolysis by directly repressing the overall gene program of the glycolytic pathway [11]. Not surprisingly, we have shown that drug resistant PD ALL cells, characterized by reduced expression of BCL6, demonstrate increased glycolysis coincident with upregulation of several molecules that modulate the metabolic pathway, including hexokinase II [9,10]. Based on these observations we screened for drugs that induce death in leukemic cells with diminished BCL6, with the intent to identify agents that could be tested for efficacy in targeting MRD in ALL. In the present study, we have successfully screened a library of FDA-approved oncology drugs in a BCL6 knockdown ALL cell line and identified cabazitaxel (CAB) and plicamycin (PLI) as potential candidates that could target and eliminate drug resistant leukemic cells. We further validated the anti-leukemic activity of CAB and PLI in six ALL cell Foliglurax monohydrochloride lines and demonstrated that part of the anti-leukemic activity was attributed to cell cycle arrest. Furthermore, to show activity in low expressing BCL6 cells, we demonstrated synergism of Foliglurax monohydrochloride the CAB/PLI combination in a cytarabine resistant REH cell line Rabbit Polyclonal to JAK2 (REH/Ara-C) and our co-culture model. Collectively our observations suggest this combination therapy, with inhibition of chemotaxis and downstream modulation of SOX2 and Mcl-1, warrants further evaluation in settings that are refractory to traditional chemotherapy. 2.?Materials and methods 2.1. Cell culture and chemicals The development of doxycycline-inducible REH BCL6 knockdown cells and its comparative REH scrambled stable cells has been previously published [10]. SUPB15 (ATCC #CRL-1929) and JM1 (ATCC #CRL-10423) were purchased and maintained in RPMI 1640 containing 10% FBS, 0.05 mM -mercapto-ethanol and 1X streptomycin/penicillin antibiotics. REH (ATCC #CRL-8286), NALM1 (ATCC #CRL-1567), NALM6 (DSMZ ACC #128), BV173 (DSMZ ACC#20), RS4 (ATCC #CRL-1873) and SD1 (DSMZ ACC#366) were purchased and maintained in RPMI 1640 containing 10% FBS and 1X streptomycin/penicillin antibiotics. Human osteoblasts (HOB) was purchased from PromoCell (Cat No: C-12720, Hiedelberg, Germany) and cultured according to the vendors recommendations. All the ALL cell lines were authenticated by short tandem repeat (STR) analysis (University of Arizona Genetic Core,.