Bortezomib- and carfilzomib-resistant myeloma cells show increased activity of all three arms of the unfolded protein response

Proteasome inhibitors are among the most potent classes of drugs in multiple myeloma treatment. One of the main challenges in myeloma therapy is acquired resistance to drugs. Several theories have been proposed to describe the mechanisms responsible for resistance to the most commonly used proteasome inhibitors bortezomib and carfilzomib. This study aimed to describe functional differences between sensitive myeloma cells (MM1S WT) and their daughter cell lines resistant to either bortezomib (MM1S/R BTZ) or carfilzomib (MM1S/R CFZ), as well as between both resistant cell lines. Bortezomib- and carfilzomib-resistant cell lines were successfully generated by continuous exposure to the drugs. When exposed to different drugs than during the resistance generation period, MM1S/R BTZ cells showed cross-resistance to carfilzomib, whereas MM1S/R CFZ cells were similarly sensitive to bortezomib as MM1S WT cells. Following proteomic profiling, unsupervised principal component analysis revealed that the MM1S/R BTZ and MM1S/R CFZ cell lines differed significantly from the MM1S WT cell line and from each other. Canonical pathway analysis showed similar pathways enriched in both comparisons - MM1S WT vs. MM1S/R CFZ and MM1S WT vs. MM1S/R BTZ. However, important differences were present in the statistical significance of particular pathways. Key alterations included the ubiquitin-proteasome system, metabolic pathways responsible for redox homeostasis and the unfolded protein response. In functional studies, both drugs continued to reduce chymotrypsin-like proteasome activity in resistant cells. However, the baseline activity of all three catalytic domains of the proteasome was higher in the resistant cells. Differences in generation of reactive oxygen species were identified in MM1S/R BTZ (decreased) and MM1S/CFZ cells (increased) in comparison to MM1S WT cells. Both baseline and drug-induced activity of the unfolded protein response were higher in resistant cells than in MM1S WT cells and included all three arms of this pathway: IRE1α/XBP1s, ATF6 and EIF2α/ATF4 (downstream effectors of PERK). In conclusion, contrary to some previous reports, resistant MM1S cells show upregulation of unfolded protein response activity, reflecting the heterogeneity of multiple myeloma and prompting further studies on the role of this pathway in resistance to proteasome inhibitors.     

Nelfinavir augments proteasome inhibition by bortezomib in myeloma cells and overcomes bortezomib and carfilzomib resistance

HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment. HIV-PI have antitumor activity via induction of ER-stress, inhibition of phospho-AKT (p-AKT) and the proteasome, suggesting antimyeloma activity. We characterize the effects of all approved HIV-PI on myeloma cells. HIV-PI were compared regarding cytotoxicity, proteasome activity, ER-stress induction and AKT phosphorylation using myeloma cells in vitro. Nelfinavir is the HIV-PI with highest cytotoxic activity against primary myeloma cells and with an IC₅₀ near therapeutic drug blood levels (8–14 μM), irrespective of bortezomib sensitivity. Only nelfinavir inhibited intracellular proteasome activity in situ at drug concentrations <40 μℳ. Ritonavir, saquinavir and lopinavir inhibited p-AKT comparable to nelfinavir, and showed similar synergistic cytotoxicity with bortezomib against bortezomib-sensitive cells. Nelfinavir had superior synergistic activity with bortezomib/carfilzomib in particular against bortezomib/carfilzomib-resistant myeloma cells. It inhibited not only the proteasomal β1/β5 active sites, similar to bortezomib/carfilzomib, but in addition the β2 proteasome activity not targeted by bortezomib/carfilzomib. Additional inhibition of β2 proteasome activity is known to sensitize cells for bortezomib and carfilzomib. Nelfinavir has unique proteasome inhibiting activity in particular on the bortezomib/carfilzomib-insensitive tryptic (β2) proteasome activity in intact myeloma cells, and is active against bortezomib/carfilzomib-resistant myeloma cells in vitro.     

Montelukast enhances cytocidal effects of carfilzomib in multiple myeloma by inhibiting mTOR pathway

Montelukast is an anti-asthmatic medication, and has recently showed its inhibitory effects on the proliferation of cancers. The purpose of this study was to identify the cytotoxic effects of montelukast on multiple myeloma (MM) cells and the combination effects of montelukast and carfilzomib in the treatment of MM. Results revealed that montelukast induced a dose- and time-dependent cytotoxicity in MM cells lines and significantly suppressed the colony formation of myeloma cells. Furthermore, montelukast enhanced the cytotoxicity of carfilzomib in MM cell lines. This anti-tumor effect was associated with decreased c-Myc via the inhibition of mTOR signaling pathway. Moreover, the combination of montelukast and carfilzomib induced apoptosis of myeloma cells effectively, even in the presence of bone marrow stromal cells (BMSCs). It is more important to note that the co-treatment exhibited similar cytocidal effects in carfilzomib-resistant cell lines (U266R and 8226R). In addition, the combined effects were noted in two MM xenograft mice models and 7 cases of human CD138⁺ myeloma cells (4 newly diagnosed cases and 3 relapsed cases) with no cytotoxicity on peripheral blood mononuclear cells (PBMCs) from 5 healthy donors. Our data suggested that montelukast enhanced the cytotoxicity of carfilzomib in both carfilzomib-sensitive and carfilzomib-resistant MM cell lines. These findings may facilitate the development of therapeutic strategies and provide a promising therapeutic combination regimen for the treatment of refractory myeloma.     

Carfilzomib and oprozomib synergize with histone deacetylase inhibitors in head and neck squamous cell carcinoma models of acquired resistance to proteasome inhibitors

Acquired resistance to proteasome inhibitors represents a considerable impediment to their effective clinical application. Carfilzomib and its orally bioavailable structural analog oprozomib are second-generation, highly-selective, proteasome inhibitors. However, the mechanisms of acquired resistance to carfilzomib and oprozomib are incompletely understood, and effective strategies for overcoming this resistance are needed. Here, we developed models of acquired resistance to carfilzomib in two head and neck squamous cell carcinoma cell lines, UMSCC-1 and Cal33, through gradual exposure to increasing drug concentrations. The resistant lines R-UMSCC-1 and R-Cal33 demonstrated 205- and 64-fold resistance, respectively, relative to the parental lines. Similarly, a high level of cross-resistance to oprozomib, as well as paclitaxel, was observed, whereas only moderate resistance to bortezomib (8- to 29-fold), and low level resistance to cisplatin (1.5- to 5-fold) was seen. Synergistic induction of apoptosis signaling and cell death, and inhibition of colony formation followed co-treatment of acquired resistance models with carfilzomib and the histone deacetylase inhibitor (HDACi) vorinostat. Synergism was also seen with other combinations, including oprozomib plus vorinostat, or carfilzomib plus the HDACi entinostat. Synergism was accompanied by upregulation of proapoptotic Bik, and suppression of Bik attenuated the synergy. The acquired resistance models also exhibited elevated levels of MDR-1/P-gp. Inhibition of MDR-1/P-gp with reversin 121 partially overcame carfilzomib resistance in R-UMSCC-1 and R-Cal33 cells. Collectively, these studies indicate that combining carfilzomib or oprozomib with HDAC or MDR-1/P-gp inhibitors may be a useful strategy for overcoming acquired resistance to these proteasome inhibitors.     

Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

Carfilzomib (Kyprolis®), a second generation proteasome inhibitor, is FDA approved for single-agent use among relapsed/refractory multiple myeloma (MM). To enhance the therapeutic efficacy of carfilzomib, we sought to combine carfilzomib with other novel agents. TG02, a multi-kinase inhibitor, targets JAK2 and CDK9. The rationale for co-treatment with carfilzomib and TG02 is that both independently target Mcl-1 and most myeloma cells are dependent on this anti-apoptotic protein for survival. We observed at least additive effects using the combination treatment in MM cell lines and patient samples. To determine how the bone marrow environment affects the efficacy of the combination we conducted co-culture experiments with Hs-5 stromal cells. We also examined the mechanism of increased apoptosis by determining the affect on expression of the Bcl-2 family of proteins. We found that carfilzomib increases NOXA mRNA expression, as expected, and TG02 treatment caused a decrease in Mcl-1 protein but not mRNA levels. Consistent with this possibility, we find silencing CDK9 does not change carfilzomib sensitivity in the same manner as addition of TG02. Since changes in Mcl-1 protein occur in the presence of a proteasome inhibitor we hypothesize that regulation of Mcl-1 translation is the most likely mechanism. Taken together our data suggest that dual inhibition of Mcl-1 via decreased expression and the induction of its antagonist NOXA by the combination of carfilzomib and TG02 is active in myeloma and warrants further testing preclinically and in clinical trials. Moreover, regulation of Mcl-1 by TG02 is more complex than initially appreciated.     

Resensitising proteasome inhibitor-resistant myeloma with sphingosine kinase 2 inhibition

The introduction of the proteasome inhibitor bortezomib into treatment regimens for myeloma has led to substantial improvement in patient survival. However, whilst bortezomib elicits initial responses in many myeloma patients, this haematological malignancy remains incurable due to the development of acquired bortezomib resistance. With other patients presenting with disease that is intrinsically bortezomib resistant, it is clear that new therapeutic approaches are desperately required to target bortezomib-resistant myeloma. We have previously shown that targeting sphingolipid metabolism with the sphingosine kinase 2 (SK2) inhibitor K145 in combination with bortezomib induces synergistic death of bortezomib-naïve myeloma. In the current study, we have demonstrated that targeting sphingolipid metabolism with K145 synergises with bortezomib and effectively resensitises bortezomib-resistant myeloma to this proteasome inhibitor. Notably, these effects were dependent on enhanced activation of the unfolded protein response, and were observed in numerous separate myeloma models that appear to have different mechanisms of bortezomib resistance, including a new bortezomib-resistant myeloma model we describe which possesses a clinically relevant proteasome mutation. Furthermore, K145 also displayed synergy with the next-generation proteasome inhibitor carfilzomib in bortezomib-resistant and carfilzomib-resistant myeloma cells. Together, these findings indicate that targeting sphingolipid metabolism via SK2 inhibition may be effective in combination with a broad spectrum of proteasome inhibitors in the proteasome inhibitor resistant setting, and is an approach worth clinical exploration.     

Downregulation of PA28α induces proteasome remodeling and results in resistance to proteasome inhibitors in multiple myeloma

Protein homeostasis is critical for maintaining eukaryotic cell function as well as responses to intrinsic and extrinsic stress. The proteasome is a major portion of the proteolytic machinery in mammalian cells and plays an important role in protein homeostasis. Multiple myeloma (MM) is a plasma cell malignancy with high production of immunoglobulins and is especially sensitive to treatments that impact protein catabolism. Therapeutic agents such as proteasome inhibitors have demonstrated significant benefit for myeloma patients in all treatment phases. Here, we demonstrate that the 11S proteasome activator PA28α is upregulated in MM cells and is key for myeloma cell growth and proliferation. PA28α also regulates MM cell sensitivity to proteasome inhibitors. Downregulation of PA28α inhibits both proteasomal load and activity, resulting in a change in protein homeostasis less dependent on the proteasome and leads to cell resistance to proteasome inhibitors. Thus, our findings suggest an important role of PA28α in MM biology, and also provides a new approach for targeting the ubiquitin-proteasome system and ultimately sensitivity to proteasome inhibitors.Subject terms: Myeloma, Translational research     

Ritonavir,nelfinavir, saquinavir and lopinavir induce proteotoxic stress in acute myeloid leukemia cells and sensitize them for proteasome inhibitor treatment at low micromolar drug concentrations

Background

Protein metabolism is an innovative potential therapeutic target for AML. Proteotoxic stress (PS) sensitizes malignant cells for proteasome inhibitor treatment. Some HIV protease inhibitors (HIV-PI) induce PS and may therefore be combined with proteasome inhibitors to achieve PS-targeted therapy of AML.

Methods

We investigated the effects of all nine approved HIV-PI alone and in combination with proteasome inhibitors on AML cell lines and primary cells in vitro.

Results

Ritonavir induced cytotoxicity and PS at clinically achievable concentrations, and induced synergistic PS-triggered apoptosis with bortezomib. Saquinavir, nelfinavir and lopinavir were likewise cytotoxic against primary AML cells, triggered PS-induced apoptosis, inhibited AKT-phosphorylation and showed synergistic cytotoxicity with bortezomib and carfilzomib at low micromolar concentrations. Exclusively nelfinavir inhibited intracellular proteasome activity, including the β2 proteasome activity that is not targeted by bortezomib/carfilzomib.

Conclusions

Of the nine currently approved HIV-PI, ritonavir, saquinavir, nelfinavir and lopinavir can sensitize AML primary cells for proteasome inhibitor treatment at low micromolar concentrations and may therefore be tested clinically toward a proteotoxic stress targeted therapy of AML.     

Increased resistance to proteasome inhibitors in multiple myeloma mediated by cIAP2 - implications for a combinatorial treatment

Despite the introduction of new treatment options for multiple myeloma (MM), a majority of patients relapse due to the development of resistance. Unraveling new mechanisms underlying resistance could lead to identification of possible targets for combinatorial treatment. Using TRAF3 deleted/mutated MM cell lines, we evaluated the role of the cellular inhibitor of apoptosis 2 (cIAP2) in drug resistance and uncovered the plausible mechanisms underlying this resistance and possible strategies to overcome this by combinatorial treatment. In MM, cIAP2 is part of the gene signature of aberrant NF-κB signaling and is heterogeneously expressed amongst MM patients. In cIAP2 overexpressing cells a decreased sensitivity to the proteasome inhibitors bortezomib, MG132 and carfilzomib was observed. Gene expression analysis revealed that 440 genes were differentially expressed due to cIAP2 overexpression. Importantly, the data imply that cIAPs are rational targets for combinatorial treatment in the population of MM with deleted/mutated TRAF3. Indeed, we found that treatment with the IAP inhibitor AT-406 enhanced the anti-MM effect of bortezomib in the investigated cell lines. Taken together, our results show that cIAP2 is an important factor mediating bortezomib resistance in MM cells harboring TRAF3 deletion/mutation and therefore should be considered as a target for combinatorial treatment.     

Effects of a novel proteasome inhibitor BU-32 on multiple myeloma cells

Proteasome inhibition is associated with substantial antitumor effects in preclinical models of multiple myeloma (MM) as well as in patients. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade^®, also called PS-341) in MM patients have shown limited activity when used as a single agent. This underscores the need to find new efficacious and less toxic proteasome inhibitors. Recently, carfilzomib was approved for the treatment of refractory/relapsed MM and several new agents have been introduced into the clinic, including marizomib and MLN9708, and trials investigating these second-generation proteasome inhibitors have demonstrated promising results. We have recently synthesized a novel proteasome inhibitor, BU-32, and tested its growth inhibitory effects in different human MM cells including RPMI8226, MM.1S, MM.1R, and U266. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499) using an in vitro MM model. BU-32 exhibits strong cytotoxicity in a panel of MM cell lines—RPMI8226, MM1S, MM1R, and U266. In addition, we demonstrate by proteasome inhibition assay that BU-32 potently inhibits the chymotryptic- and caspase-like activities of the 26S proteasome. We further show from Annexin V-FITC binding studies that BU-32, like Bortezomib, induces apoptosis in a panel of MM cell lines but the effect is more pronounced with BU-32-treated cells. Invasion assay with the MM.1S cell line indicates that BU-32 inhibits the invasiveness of myeloma cells. Results from our studies using real-time PCR array analyses show that BU-32 effectively downregulates an array of angiogenesis and inflammatory markers. Our results suggest that BU-32 might be a potential chemotherapeutic agent with promising antitumor activity for the treatment of MM.     

Targeting Mcl-1 for multiple myeloma (MM) therapy: Drug-induced generation of Mcl-1 fragment Mcl-1 triggers MM cell death via c-Jun upregulation

Myeloid cell leukemia-1 (Mcl-1, HGNC: 6943), a pro-survival member of the Bcl-2 family, plays a crucial role in Multiple Myeloma (MM) pathogenesis and drug resistance, thus representing a promising therapeutic target in MM. A novel strategy to inhibit Mcl-1 activity is the induction of ubiquitin-independent Mcl-1 degradation. Our own and other previous studies have demonstrated caspase-dependent generation of a 28 kDa Mcl-1 fragment, Mcl-1^128–350, which inhibits MM cell proliferation and survival. Here, we show that similar to bortezomib, the novel proteasome inhibitors carfilzomib and ixazomib, as well as staurosporine and adaphostin, induce the generation of Mcl-1^128–350 in MM cells. Next, the molecular sequelae downstream of Mcl-1^128–350, which mediate its pro-apoptotic activity, were delineated. Surprisingly, we observed nuclear accumulation of drug-induced or exogenously overexpressed Mcl-1^128–350, followed by elevated mRNA and protein levels of c-Jun, as well as enhanced AP-1 reporter activity. Moreover, drug-induced AP-1 activity was blocked after introducing a point mutation into the highly conserved Mcl-1 caspase-cleavage site Asp127, but not Asp157. Consequently, drug-triggered cell death was significantly decreased in MM cells transfected with Mcl-1 D127A, but not with Mcl-1 D157A. Consistent with these data, treatment with bortezomib triggered c-Jun upregulation followed by apoptosis in Mcl-1^wt/wt, but not Mcl-1^Δ/null murine embryonic fibroblasts (MEFs). Transfection of a plasmid carrying Mcl-1^wt into Mcl-1^Δ/null MEFs restored bortezomib-induced Mcl-1 fragmentation, c-Jun upregulation and AP-1 reporter activity. Finally, our data indicate that drug-induced generation of a pro-apoptotic Mcl-1 fragment followed by c-Jun upregulation may also be a novel therapeutic approach in other tumor entities.     

Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth

Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein.     

The 39th David A. Karnofsky Lecture: bench-to-bedside translation of targeted therapies in multiple myeloma

Multiple myeloma (MM) is a remarkable example of rapid bench-to-bedside translation in new drug development. The proteasome inhibitor bortezomib and immunomodulatory drug lenalidomide targeted MM cells in the bone marrow (BM) microenvironment to overcome conventional drug resistance in laboratory and animal models and were rapidly translated into clinical trials demonstrating their efficacy in patients with relapsed and then newly diagnosed MM, with a doubling of the median survival as a direct result. The future is even brighter. First, immune-based therapies are being developed (eg, elotuzumab monoclonal antibody [MoAb]; CD138DM immunotoxin; MM cell-dendritic cell vaccines; CD138, CS-1, and XBP-1 peptide vaccines; anti-17 MoAb; and other treatments to overcome causes of immune dysfunction). Second, promising next-generation agents target the MM cell in its microenvironment (eg, deubiquitinating enzyme inhibitors; chymotryptic [carfilzomib, Onyx 0912, MLN 9708] and broader [NPI-0052] proteasome inhibitors; immunoproteasome inhibitors; and pomalidamide). Moreover, agents targeting bone biology (eg, zoledronic acid, anti-DKK-1 MoAb, anti-B-cell activating factor MoAb and bortezomib, Btk inhibitor) show promise not only in preserving bone integrity but also against MM. Third, rationally based combination therapies, including bortezomib with Akt, mammalian target of rapamycin, or histone deacetylase inhibitors, are active even in bortezomib-refractory MM. Finally, genomics is currently being used in the definition of MM heterogeneity, new target discovery, and development of personalized therapy. Myeloma therefore represents a paradigm for targeting the tumor in its microenvironment, which has already markedly improved patient outcome in MM and has great potential in other hematologic malignancies and solid tumors as well.     

Molecular chaperone GRP78 enhances aggresome delivery to autophagosomes to promote drug resistance in multiple myeloma

Despite the clinical benefit of the proteasome inhibitor bortezomib, multiple myeloma (MM) patients invariably relapse through poorly defined mechanisms. Myeloma cells inevitably develop chemoresistance that leads to disease relapse and patient-related deaths. Studies in tumor cell lines and biopsies obtained from patients refractory to therapy have revealed that myeloma cells adapt to stress by inducing expression of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone with anti-apoptotic properties. Treatment of myeloma cells with bortezomib increased GRP78 levels and activated GRP78-dependent autophagy. Expression profiling indicated that GRP78-encoding HSPA5 was significantly upregulated in bortezomib-resistant cells. Co-treatment with the anti-diabetic agent metformin suppressed GRP78 and enhanced the anti-proliferative effect of bortezomib. Bortezomib treatment led to GRP78 co-localization with proteotoxic protein aggregates, known as aggresomes. Pharmacologic suppression, genetic ablation or mutational inactivation of GRP78 followed by bortezomib treatment led to the accumulation of aggresomes but impaired autophagy and enhanced anti-myeloma effect of bortezomib. GRP78 was co-immunoprecipitated with the KDEL receptor, an ER quality control regulator that binds proteins bearing the KDEL motif to mediate their retrieval from the Golgi complex back to the ER. Taken together, we demonstrate that inhibition of GRP78 functional activity disrupts autophagy and enhances the anti-myeloma effect of bortezomib.     

Targeting autophagy reverses de novo resistance in homologous recombination repair proficient breast cancers to PARP inhibition

Background Poly(ADP-ribose) polymerase inhibitors (PARPi) target tumours defective in homologous recombination (HR). Most BRCA-wild-type (WT) HR-proficient breast cancers are intrinsically resistant to PARP inhibitors, e.g., talazoparib. We evaluated the role of autophagy in this de novo resistance and determined the underlying mechanism to overcome this.Methods Autophagosome formation and autophagic flux were assessed by evaluating endogenous LC3-II levels and ectopic expression of EGFP-LC3 and mRFP-EGFP-LC3 in breast cancer cells. Autophagy-defective cells were generated by genetic depletion of BECN1, ATG5, p62/SQSTM1 and LAMP1 by using CRISPR-Cas9 double nickase system. The response of PARPi was evaluated in autophagy-proficient and -defective breast cancer cells and in xenograft SCID-mice model.Results Pro-survival autophagy was significantly enhanced upon talazoparib treatment in BRCA-WT breast cancer cell lines. Autophagy-deficient cells were hypersensitive to talazoparib. Targeting autophagy synergistically enhanced the therapeutic efficacy of talazoparib in BRCA1-WT breast cancer cells in vitro and in vivo xenograft tumour mouse model. Mechanistically, autophagy inhibition by chloroquine promoted deleterious NHEJ mediated DSB-repair, leading to extensive genomic instability and mitotic catastrophe.Conclusions Autophagy confers de novo resistance to PARP inhibitor, talazoparib. Autophagy inhibition improves the therapeutic outcome of PARPi treatment in preclinical mice model, bearing HR-proficient breast tumours, warranting its usage in the clinical settings.Subject terms: Breast cancer, Cancer therapy     

Krüppel-like factor 9 upregulates E-cadherin transcription and represses breast cancer invasion and metastasis

Aberrant expression of Krüppel-like factor 9 (KLF9) is frequently found in some types of cancer and is implicated in cancer initiation and progression. However, the effects of KLF9 on cancer metastases and the underlying mechanisms still need to be understood. Here, we found that KLF9 evidently inhibited the capabilities of migration and invasion of breast cancer cells. The expression of KLF9 was markedly decreased in breast cancer patients compared with benign tumors, and was positively correlated with the expression of E-cadherin in the tissues of breast cancer patients. Mechanistically, chromatin immunoprecipitation combined with site-directed mutagenesis-luciferase assay revealed that KLF9 activated the E-cadherin promoter by binding to GT-box elements located +84 bp and -143 bp from the TSS in the E-cadherin promoter, leading to elevated expression of E-cadherin mRNA and protein. In vivo experiments confirmed that KLF9 strongly inhibited the lung metastasis of breast cancer and increased mouse E-cadherin expression in 4T1 mouse breast cancer cells. Taken together, our findings demonstrated that KLF9 could suppress breast cancer invasion and metastasis by upregulating E-cadherin, which provided new insight into aggressive treatment of breast cancer by targeting the KLF9/E-cadherin axis.     

Histone Deacetylase Inhibitors in Multiple Myeloma: Rationale and Evidence for Their Use in Combination Therapy

Multiple myeloma (MM) arises from abnormal proliferation and survival (ie, a high proliferative index and a low apoptotic index) of mature immunoglobulin-producing plasma cells in the bone marrow. Development of novel therapeutic options, such as proteasome inhibitors and immunomodulatory agents (IMiDs), has improved treatment outcomes. However, patients often develop relapsed and refractory MM, thus requiring alternative treatment approaches. Histone acetyltransferases and histone deacetylases (HDACs) control the acetylation status of proteins and affect a broad array of physiologic processes (eg, cell cycle, apoptosis, and protein folding) involved in cell growth and survival. The discovery that HDACs might have a role in various hematologic malignancies, including MM, has led to the development of HDAC inhibitors as potential antitumor agents. Preclinical evidence from studies of HDAC inhibitors in combination with proteasome inhibitors (eg, bortezomib and carfilzomib), other antimyeloma agents, including IMiDs (eg, lenalidomide), and cytotoxic agents (eg, melphalan, pegylated liposomal doxorubicin), provides a strong scientific rationale for the evaluation of these regimens. Results from early stage clinical trials further support the use of HDAC inhibitors as a therapeutic option for MM, in combination with current and emerging antimyeloma agents. In this review, we examine the role of protein acetylation that underlies the antimyeloma effects of HDAC inhibitors, discuss the preclinical rationale for the use of HDAC inhibitors in combination with other antimyeloma agents, and provide an overview of the current clinical evidence supporting the use of HDAC inhibitors as a therapeutic option in MM.