Figure 3.
Class IIa HDAC inhibition increases transcription of BCMA. (A) Volcano plot indicating the BCMA expression phenotype and statistical significance for knockdown (CRISPRi) of human genes (orange dots) and quasi-genes generated from negative control sgRNA (gray dots). Genes in the HDAC family are shown as green dots and labeled with the HDAC number. (B) RPMI8226 cells were treated with increasing concentrations of the pan-HDAC inhibitor panobinostat (10 nM, 25 nM), the HDAC6-specific inhibitor ricolinostat (0.5 μM, 1 μM), the class II HDAC inhibitor TMP269 (5 μM, 10 μM), or DMSO for 48 hours and analyzed by using flow cytometry for cell surface expression of BCMA and CD38. Fold changes in protein levels were determined by normalizing to the DMSO-treated cells. Data points are means of 3 biological replicates, and error bars denote standard deviations. (C-D) Total protein extracts from RPMI8226 cells treated with 2.5 μM, 5 μM, and 10 μM of TMP269 for 48 hours were analyzed by using immunoblotting for expression levels of BCMA. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used to normalize differences in loading amounts. Data are represented as fold change relative to the total protein expression level after normalization with GAPDH. Data points are means of 2 technical replicates, and error bars denote standard deviations. (E) RPMI8226 cells treated with 10 nM of panobinostat, 0.5 μM of ricolinostat, and 5 μM and 10 μM of TMP269 for 48 hours were processed for quantitative polymerase chain reaction to determine transcript levels of BCMA and CD38. Fold changes in transcript levels with different drug treatments were determined after normalizing to the β-actin gene. Data are means of 2 biological replicates, and error bars denote standard deviations. (F) RPMI8226 cells were treated with DMSO, 5 μM and 10 μM of TMP269, and 5 μM of the γ-secretase inhibitor RO4929097 as single agents or in combination for 48 hours and analyzed by using flow cytometry for cell surface expression of BCMA. Fold changes in protein levels were determined by normalizing to the DMSO-treated cells. Data points are means of 3 biological replicates, and error bars denote standard deviations.

Class IIa HDAC inhibition increases transcription of BCMA. (A) Volcano plot indicating the BCMA expression phenotype and statistical significance for knockdown (CRISPRi) of human genes (orange dots) and quasi-genes generated from negative control sgRNA (gray dots). Genes in the HDAC family are shown as green dots and labeled with the HDAC number. (B) RPMI8226 cells were treated with increasing concentrations of the pan-HDAC inhibitor panobinostat (10 nM, 25 nM), the HDAC6-specific inhibitor ricolinostat (0.5 μM, 1 μM), the class II HDAC inhibitor TMP269 (5 μM, 10 μM), or DMSO for 48 hours and analyzed by using flow cytometry for cell surface expression of BCMA and CD38. Fold changes in protein levels were determined by normalizing to the DMSO-treated cells. Data points are means of 3 biological replicates, and error bars denote standard deviations. (C-D) Total protein extracts from RPMI8226 cells treated with 2.5 μM, 5 μM, and 10 μM of TMP269 for 48 hours were analyzed by using immunoblotting for expression levels of BCMA. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used to normalize differences in loading amounts. Data are represented as fold change relative to the total protein expression level after normalization with GAPDH. Data points are means of 2 technical replicates, and error bars denote standard deviations. (E) RPMI8226 cells treated with 10 nM of panobinostat, 0.5 μM of ricolinostat, and 5 μM and 10 μM of TMP269 for 48 hours were processed for quantitative polymerase chain reaction to determine transcript levels of BCMA and CD38. Fold changes in transcript levels with different drug treatments were determined after normalizing to the β-actin gene. Data are means of 2 biological replicates, and error bars denote standard deviations. (F) RPMI8226 cells were treated with DMSO, 5 μM and 10 μM of TMP269, and 5 μM of the γ-secretase inhibitor RO4929097 as single agents or in combination for 48 hours and analyzed by using flow cytometry for cell surface expression of BCMA. Fold changes in protein levels were determined by normalizing to the DMSO-treated cells. Data points are means of 3 biological replicates, and error bars denote standard deviations.

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