IKZF1/3 and CRL4-CRBN E3 ubiquitin ligase mutations and IMiD resistance in multiple myeloma

The work was supported by the Deutsche Forschungsgemeinschaft (KFO216), the IZKF, the BTHA and the CDW Stiftung (KMK). UM was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg.S

Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation - An Appraisal

Cell lines represent the everyday workhorses for in vitro research on multiple myeloma (MM) and are regularly employed in all aspects of molecular and pharmacological investigations. Although loss-of-function studies using RNA interference in MM cell lines depend on successful knockdown, no well-established and widely applied protocol for efficient transient transfection has so far emerged. Here, we provide an appraisal of electroporation as a means to introduce either short-hairpin RNA expression vectors or synthesised siRNAs into MM cells. We found that electroporation using siRNAs was much more efficient than previously anticipated on the basis of transfection efficiencies deduced from EGFP-expression off protein expression vectors. Such knowledge can even confidently be exploited in "hard-to-transfect" MM cell lines to generate large numbers of transient knockdown phenotype MM cells. In addition, special attention was given to developing a protocol that provides easy implementation, good reproducibility and manageable experimental costs

Voltage dependence of electroporation and knockdown efficiency in AMO-1 cells.

<p>Transfection of AMO-1 cells across a range of voltages using an expression vector for EGFP (pEGFP-N3) and a stealth siRNA against ERK2 (stERK2) in the electroporation mixture. Top panel: increases of the fractions of EGFP-expressing as well as of dead cells with higher voltages (top row). Cells taken in culture after OptiPrep-mediated debris removal reflect only the increase in transfection efficiency for the EGFP expression plasmid (bottom row). Middle panel: purified AMO-1 cells (those shown in the upper panel, bottom row) after culture for another 4 days. Top row: EGFP expression. Bottom row: annexin V-PromoFluor 647/PI staining. Even for the highest voltage used (320 V) the purified live cell fraction did not fare worse in subsequent culture than cells electroporated under milder conditions. Bottom panel: Western analysis of ERK2 knockdown at days 3 and 5 post-electroporation from the same cultures from which the FACS panels were derived. Efficient siRNA-mediated ERK2 knockdown was achieved at voltages significantly lower than required for the best levels of plasmid electroporation. However, a lower limit for successful knockdown was reached between the settings for 160 and 200 V. Shown is a representative experiment of two complete sets (Western blotting included). Anti-ERK1/2 antibody: CST.</p

Electroporation of INA-6 cells stably expressing enhanced green fluorescent protein with an siRNA oligonucleotide against EGFP.

<p>INA-6-EGFP cells were electroporated with a solution containing a stealth siRNA targeting EGFP as well as an expression plasmid for CD4Δ. One day post-electroporation one half of the cell culture was purified according to the column procedure (red curves, also see Fig. 1b)–e)), whereas the other half only underwent debris removal with OptiPrep (blue curves, also see Fig. 1f)). Purified cells were further cultured and FACS-analysed for EGFP expression at the times indicated. Only the live cell fraction (as demarcated in the forward/sideward scatter) was analysed and plotted against similarly treated INA-6-EGFP cells (green curves) transfected with a non-EGFP targeting siRNA. Knockdown efficiency was essentially identical in strength and over time between both purification approaches. One representative experiment from a total of three is shown.</p

Knockdown efficiency in MM cells.

<p>Knockdown of ERK2 in different MM cell lines after transfection with either a short-hairpin expression vector (pSU-ERK2) or the “corresponding” target sequence synthesised as 25 bp stealth siRNA (stERK2; see Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097443#pone.0097443-Chatterjee1" target="_blank">[21]</a>). At day 1 post-electroporation half of the transfected culture was subjected to the column purification method (also see Fig. 1b)–e)) (resp. cell sorting for AMO-1 cells) and the other half to debris removal only (also see Fig. 1f)). Cells were harvested for Western blotting at the times indicated. Empty pSUPER vector (pSU) transfected cells served as controls. The blots show that the ERK2 knockdown efficiency for stealth siRNA is virtually identical between cells that only underwent debris removal and those that were subjected to the column purification procedure. ERK2 knockdown using the short-hairpin expression vector was less efficient in debris-removal-only samples compared with their cognate column purification complements (see JJN-3, L-363). Representative experiments (JJN-3: n = 3; L-363: n = 2, AMO-1: n = 2) are shown. Anti-ERK1/2 antibody: CST.</p

Electroporation of AMO-1 cells with a 6-FAM-labelled siRNA oligonucleotide.

<p>Left column: Fluorescence of AMO-1 cells electroporated with the siERK2-6-FAM oligonucleotide (green curve) in relation to mock transfected cells (blue curve) at different time points post-electroporation. Right: Western analysis for ERK2 knockdown at days 3, 5, 7 post-electroporation. One representative experiment from a total of three is shown. Anti-ERK1/2 antibody: CST.</p

Electroporation and knockdown efficiencies in “easy-to-transfect” vs. “hard-to-transfect” MM cell lines.

<p>Left-hand panel: MM cell lines were electroporated with an expression vector for EGFP (pEGFP-N3; 10 µg/ml) and stealth siRNAs against either ERK2 (stERK2; 3 µM) or against no specific target (control; 3 µM). The FACS-measurements represent the cell cultures at day 1 post-electroporation after debris removal with OptiPrep. Right-hand panel: Knockdown of ERK2 and intrinsic levels of phospho-ERK2 (cells from the cultures represented on the left were harvested at day 3 post-electroporation for Western blotting). Good knockdown of ERK2 and lowered levels of phospho-ERK2 were found for all four MM cell lines tested. Shown is a representative experiment of two complete sets (Western blotting included). Anti-ERK1/2 antibody: Santa Cruz Biotechnology.</p

Electroporation of MM cell lines and subsequent purification of transfected cells.

<p>Shown is a representative example of the procedure using the well-transfectable MM cell line JJN-3. This standard column purification has now been performed hundreds of times in our laboratory and is also easily applicable for MM cell lines INA-6, KMS-11, L-363, MM.1S and U-266 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097443#pone-0097443-t001" target="_blank">Table 1</a>). a) Cell culture one day after electroporation with expression plasmids for enhanced green fluorescent protein (EGFP) and CD4Δ, showing about a quarter of cells strongly positive for EGFP. b)-e) Enrichment of strongly transfected cells by selection for CD4 surface expression (CD4 MicroBead column selection). b) Column runthrough of cell culture shown in a). Of note is the similar look with a), but with depletion of the strongest transfected cells in b). c) Column eluate of the cell culture shown in a). Untransfected cells (EGFP- and CD4Δ-negative) have effectively been removed, but the column procedure tends to retain significant amounts of dead cells (EGFP-negative, PI-positive). d) Floating fraction of the column eluate as shown in c) after “density gradient” (more properly: density step) treatment using OptiPrep, consisting mostly of viable and strongly transfected cells. e) Pelleted fraction of the column eluate as shown in c) after “density gradient” treatment using OptiPrep, consisting mostly of debris. f) Removal of debris by OptiPrep treatment from the cell culture as shown in a) without prior column separation, leaving two main fractions which are either EGFP-negative, or distinctly EGFP-positive. See Methods section for further details.</p

The IKK inhibitor Bay 11-7082 induces cell death independent from inhibition of activation of NFκB transcription factors.

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells