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Letters to the Editor
2 dependency of this t(11;14) carrying cell line.3 In con- clusion, these results strengthen a model where BCL-XL is capable of sequestering BAK and BIM released by S63845, thus prohibiting the onset of the apoptotic sig- naling cascade.
We next aimed to confirm the impact of panobinostat and ricolinostat on histone and tubulin acetylation as mechanism of synergy with MCL-1 inhibitors. Panobinostat is expected to strongly increase histone acetylation, while ricolinostat, which is selectively tar- geting HDAC6,13,14 should increase acetylated α-tubulin without altering acetylated histone levels.15 We treated MM.1S, U266 and KMS-12-BM cells with both HDACi and assessed acetylated and total protein expression levels of α-tubulin and histone-3 after 24 h. Panobinostat strongly increased acetyl-histone-3 in all three cell lines (Figure 3A; Online Supplementary Figure S3E and F). Contrary to our expectations, ricolinostat treatment likewise led to a strong increase in acetyl-his- tone-3 besides elevating acetyl-α-tubulin levels (Figure 3A; Online Supplementary Figure S3E and E), indicating that ricolinostat has off-target effects on additional HDAC family members.
Accordingly, we aimed to investigate whether the synergism of ricolinostat and S63845 is facilitated via HDAC6 inhibition or via the epigenetic off-target effect. To this end, we performed viability assays with MM.1S and U266 cells transduced with a Tet-on miR-E plasmid harboring a control short hairpin RNA (shRNA) (Renilla), or HDAC6 shRNA. HDAC6 knockdown was confirmed 48 h after induction with 0.3 μg/mL doxycy- cline (Figure 3B; Online Supplementary Figure S3G). However, S63845 significantly decreased cell viability regardless of the presence of HDAC6 knockdown (Figure 3C and D; Online Supplementary Figure S3H to J). These results suggest that the synergism between S63845 and ricolinostat is due to the unspecific epige- netic effect of ricolinostat.
In order to validate our findings in primary MM cells
we treated CD138-selected MM cells ex vivo for 20 h
with single-agent HDACi and S63845 as well as the cor-
responding combinations before evaluating apoptosis
induction. This demonstrated an increase of apoptotic
cells upon combination treatment with S63845+HDACi
in three of four samples tested, whereas the magnitude
was highly variable (Figure 3E to H). Unfortunately, we
were unable to collect sufficient cell material to estab-
lish a link between BCL-2 family dependencies and
combination activity. However, we investigated
whether the synergism was accompanied by a down-
regulation of BCL-XL in MM patient samples ex vivo. For
this purpose, we treated patient samples for 10 h with
S63845 alone or in combination with HDACi and deter-
mined BCL-XL protein expression. In both analyzed
patient samples, downregulation of BCL-XL protein
expression was observed in the MM cell lines (approxi-
mately 30% vs. S63845 single-agent treatment) upon
S63845 combination with either panobinostat or ricoli-
nostat, respectively. (Figure 3I to J). This suggests that
the combination of MCL-1 inhibitors with HDACi is
capable to tackle both, MCL-1 and
BCL-X , in MM patient cells. However, our findings L
need to be confirmed in enlarged patient cohorts and advanced in vivo models (i.e., carrying humanized MCL- 1)4 to better evaluate the clinical potential of our results as well as to define patient stratification markers.
In conclusion, our findings support a model where
BCL-X sequesters BAK/BIM released in response to L
MCL-1 inhibition, particularly in tumor clones with
baseline BCL-XL functionality such as MM.1S and U266 cells. By combining MCL-1i with HDACi, BCL-XL pro- tein is downregulated, leading to unrestrained BAK acti- vation and initiation of the apoptotic signaling cascade (Figure 3K). Previous efforts to pharmacologically target BCL-XL failed due to its role in megakaryopoiesis.12,11 Hence, our findings point towards an alternative oppor- tunity to indirectly tackle BCL-XL/MCL-1 co-dependent MM cells by combining MCL-i1 and HDACi and high- light the importance of exploring various options of apoptosis induction for designing new treatment con- cepts for clinical evaluation
Anja Schneller, Niklas Zojer,* Arnold Bolomsky* and Heinz Ludwig*
*NZ, AB and HL contributed equally as co-senior authors
Department of Medicine I, Wilhelminen Cancer Research Institute, Clinic Ottakring, Vienna, Austria
Correspondence:
HEINZ LUDWIG - heinz.ludwig@extern.gesundheitsver- bund.at
doi:10.3324/haematol.2020.277152 Received: November 30, 2020.
Accepted: April 20, 2021.
Pre-published: April 29, 2021.
Disclosures: no conflicts of interest to disclose.
Contributions: AS designed and performed experiments and analyzed results; AB encouraged the investigation, designed experiments and provided intellectual input and expertise;
NZ provided supervision and expertise; HL provided supervision and secured funding; AS and AB wrote the manuscript.
Acknowledgments: the authors would like to thank Waltraud Scherbler and Martin Schreder for providing primary patient material and Anna Walzl for excellent assistance.
Funding: this study was funded by the Wilhelminen Cancer Research Institute, the Austrian Forum Against Cancer and the Austrian Academy of Science (# 25542). AS is the recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Wilhelminen Cancer Research Institute.
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