American Society of Hematology

Delineating Molecular Mechanisms of Proteasome and Histone Deacetylase Inhibitor-Induced Myeloma Cytotoxicity

Kenneth C. Anderson, MD

Published on: March 01, 2012

Dr. Anderson serves on advisory boards for Millennium and Novartis.

Mannava S, Zhuang D, Nair JR et al. KLF9 is a novel transcriptional regulator of bortezomib-and LBH589-induced apoptosis in multiple myeloma cells. Blood. 2011. Epub ahead of print.

In this study, Mannava and colleagues identified Kruppel-like family factor 9 (KLF9) as an important transcriptional regulator of apoptosis in multiple myeloma (MM) cells. Based on the observation of increased KLF9 transcript levels in tumor cells from patients with relapsed MM who responded to bortezomib, they characterized the role of KLF9 in mediating cytotoxicity. Indeed, KLF9, as well as proapoptotic Noxa, are induced in MM cells by bortezomib in association with an observed increase in acetylation of histone H3 due to inhibition of histone deacetylation. They showed that histone deacetylase inhibitor LBH589 (panobinostat) not only increased acetylated H3 and Noxa, but also increased KLF9 at both the gene and protein levels, and conversely, that knockdown of KLF9 inhibited both bortezomib- and LBH589- triggered Noxa induction and related MM cell death. In their studies, bortezomib-induced KLF9 was shown to bind directly to the Noxa promoter, and Noxa knockdown, at least in part, abrogated cytotoxicity in MM cells induced either by bortezomib or by KLF9 overexpression. Together, these studies implicate KLF9 as a transcriptional regulator of drug-induced apoptosis in MM cells.

The proteasome inhibitor bortezomib is approved to treat both relapsed/refractory and newly diagnosed MM and confers an overall survival benefit at the five-year follow-up when used as initial therapy. In preclinical studies, bortezomib effects a remarkable variety of biologic events in MM cells including the following: direct targeting of proteasome chymotrypsin- and caspase-like activities; decreased MM cell growth and survival (inhibition of NF-κB, MAPK, and Jak/STAT with activation of PI3K-Akt signaling); ER stress induction (caspase 12 cleavage, increased phospho- PERK, GADD-153, ATF4, GRP78, and XBP-1 splicing); triggering of apoptosis (increased JNK, caspases and PARP cleavage, ROS, cytochrome c and Smac release, mitochondrial Ca influx, Bid cleavage, Fas and FasL, BH3 only proteins Bim, Bik, and Noxa, along with decreased mitochondrial membrane potential and IAP proteins); induction of heat shock proteins (increased Hsp 27, 70, and 90) and inhibition of DNA repair (decreased DNA-PK); and cell-cycle inhibition (increased p21, p27, p53 with cyclins D1, E1, A, and B). Moreover, bortezomib targets the tumor cell-bone marrow microenvironment interaction (decreased expression of adhesion molecules ICAM, VCAM, and αVβ3 as well as IGF-1, IL-6, BAFF, and RANKL) and inhibits angiogenesis (decreased migration and VEGF, MMP9, and caveolin-1) and modulates bone turnover (decreased osteoclastogenesis, MIP1α, and BAFF with increased osteoblast formation). Which of these and other preclinical activities confer the observed clinical benefits remains unclear. Importantly, profiling studies of clinical samples from treated patients can give clues as to targets and mechanisms of response (i.e., NF-κB activation in tumor cells is associated with response). Here, profiling of patient samples showed that KLF9 expression identified responders, and elegant bedside-back-to-bench studies delineated the mechanism linking drug-related upregulation of KLF9 gene and protein expression to Noxa, and ultimately to MM cell apoptosis.

Prior preclinical studies in MM have provided the basis for combining bortezomib with the Akt inhibitor perifosine, as well as with histone deacetylase inhibitors panobinostat and vorinostat, ultimately leading to phase III clinical trials of these combinations. Mannava and colleagues add further mechanistic evidence underlying this synergistic activity. Bortezomib downregulates class I histone deacetylase activity, and HDAC inhibitor panobinostat further enhances histone H3 and H4 acetylation induced by bortezomib. Importantly, KLF9 upregulation by bortezomib is further enhanced by panobinostat, thereby defining the molecular mechanism of enhanced apoptosis through transcriptional enhancement of Noxa expression. These studies therefore identify KLF9 as a target for novel single-agent and combination therapies in MM.

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