Overcoming ABCG2-mediated drug resistance with imidazo-[1,2-b]-pyridazine-based Pim1 kinase inhibitors

Purpose Multidrug efflux pumps such as ABCG2 confer drug resistance to a number of cancer types, leading to poor prognosis and outcome. To date, the strategy of directly inhibiting multidrug efflux pumps in order to overcome drug resistance in cancer has been unsuccessful. An alternative strategy is to target proteins involved in the regulation of multidrug efflux pump activity or expression. Pim1 kinase has been demonstrated to phosphorylate ABCG2, promote its oligomerisation and contribute to its ability to confer drug resistance. Methods In the present manuscript, imidazo-pyridazine-based inhibitors of Pim1 were examined for their ability to overcome ABCG2-mediated drug resistance. Drug efficacy was measured as a cytotoxic response or an effect on transport by ABCG2. Protein expression patterns were assessed using western immuno-blotting. Results The two Pim1 inhibitors increased the potency of flavopiridol, mitoxantrone, topotecan and doxorubicin, specifically in ABCG2-expressing cells. This effect was associated with an increase in the cellular accumulation of [3H]-mitoxantrone, suggesting direct impairment of the transporter. However, prolonged pre-incubation with the studied inhibitors greatly enhanced the effect on mitoxantrone accumulation. The inhibitors caused a significant time-dependent reduction in the expression of ABCG2 in the resistant cells, an effect that would improve drug efficacy. Conclusion Consequently, it appears that the Pim1 inhibitors display a dual-mode effect on ABCG2-expressing cancer cells. This may provide a powerful new strategy in overcoming drug resistance by targeting proteins that regulate expression of efflux pumps

Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins

The function of small ubiquitin-like modifier (SUMO)-binding proteins is key to understanding how SUMOylation regulates cellular processes. We identified two related Schizosaccharomyces pombe proteins, Rfp1 and Rfp2, each having an N-terminal SUMO-interacting motif (SIM) and a C-terminal RING-finger domain. Genetic analysis shows that Rfp1 and Rfp2 have redundant functions; together, they are essential for cell growth and genome stability. Mammalian RNF4, an active ubiquitin E3 ligase, is an orthologue of Rfp1/Rfp2. Rfp1 and Rfp2 lack E3 activity but recruit Slx8, an active RING-finger ubiquitin ligase, through a RING–RING interaction, to form a functional E3. RNF4 complements the growth and genomic stability defects of rfp1rfp2, slx8, and rfp1rfp2slx8 mutant cells. Both the Rfp-Slx8 complex and RNF4 specifically ubiquitylate artificial SUMO-containing substrates in vitro in a SUMO binding-dependent manner. SUMOylated proteins accumulate in rfp1rfp2 double-null cells, suggesting that Rfp/Slx8 proteins may promote ubiquitin-dependent degradation of SUMOylated targets. Hence, we describe a family of SIM-containing RING-finger proteins that potentially regulates eukaryotic genome stability through linking SUMO-interaction with ubiquitin conjugation

MLL-rearranged acute lymphoblastic leukemias activate BCL-2 through H3K79 methylation and are sensitive to the BCL-2-specific antagonist ABT-199

Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias

Deubiquitinase USP13 dictates MCL1 stability and sensitivity to BH3 mimetic inhibitors

MCL1, a pro-survival BCL-2 related protein with rapid turnover rate, is often dysregulated in cancers. Here, the authors show that MCL1’s stability is regulated by deubiquitinase USP13, and its inhibition sensitises tumor cells to BH3 mimetic inhibitors