[Show abstract][Hide abstract] ABSTRACT: KDM3A is implicated in tumorigenesis; however, its biological role in multiple myeloma (MM) has not been elucidated. Here we identify KDM3A-KLF2-IRF4 axis dependence in MM. Knockdown of KDM3A is toxic to MM cells in vitro and in vivo. KDM3A maintains expression of KLF2 and IRF4 through H3K9 demethylation, and knockdown of KLF2 triggers apoptosis. Moreover, KLF2 directly activates IRF4 and IRF4 reciprocally upregulates KLF2, forming a positive autoregulatory circuit. The interaction of MM cells with bone marrow milieu mediates survival of MM cells. Importantly, silencing of KDM3A, KLF2 or IRF4 both decreases MM cell adhesion to bone marrow stromal cells and reduces MM cell homing to the bone marrow, in association with decreased ITGB7 expression in MAF-translocated MM cell lines. Our results indicate that the KDM3A-KLF2-IRF4 pathway plays an essential role in MM cell survival and homing to the bone marrow, and therefore represents a therapeutic target.
Full-text · Article · Jan 2016 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Multiple Myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response, to genomic stability and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of MAPK pathway genes, MAPK-signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1, confers resistance to DNA damage. Using genetic and biochemical studies in vitro and, in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications both for tumorigenesis and treatment of MM.
[Show abstract][Hide abstract] ABSTRACT: Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.
[Show abstract][Hide abstract] ABSTRACT: B cell malignancies frequently colonize the bone marrow. The mechanisms responsible for this preferential homing are incompletely understood. Here we studied multiple myeloma (MM) as a model of a terminally differentiated B cell malignancy that selectively colonizes the bone marrow. We found that extracellular CyPA (eCyPA), secreted by bone marrow endothelial cells (BMECs), promoted the colonization and proliferation of MM cells in an in vivo scaffold system via binding to its receptor, CD147, on MM cells. The expression and secretion of eCyPA by BMECs was enhanced by BCL9, a Wnt-β-catenin transcriptional coactivator that is selectively expressed by these cells. eCyPA levels were higher in bone marrow serum than in peripheral blood in individuals with MM, and eCyPA-CD147 blockade suppressed MM colonization and tumor growth in the in vivo scaffold system. eCyPA also promoted the migration of chronic lymphocytic leukemia and lymphoplasmacytic lymphoma cells, two other B cell malignancies that colonize the bone marrow and express CD147. These findings suggest that eCyPA-CD147 signaling promotes the bone marrow homing of B cell malignancies and offer a compelling rationale for exploring this axis as a therapeutic target for these malignancies.
[Show abstract][Hide abstract] ABSTRACT: Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.
Preview · Article · May 2015 · Blood Cancer Journal
[Show abstract][Hide abstract] ABSTRACT: Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biologic role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming ATF4 plays a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressor, including proteasome inhibitors, to improve patient outcome in MM.Leukemia accepted article preview online, 24 March 2015. doi:10.1038/leu.2015.83.
No preview · Article · Mar 2015 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: XBP1 is a critical transcriptional activator of the unfolded protein response (UPR), which increases tumor cell survival under prolonged endoplasmic reticulum (ER) stress and hypoxic conditions.This study was designed to evaluate the immunogenicity of heteroclitic XBP1 unspliced (US)184-192 (YISPWILAV) and heteroclictic XBP1 spliced (SP)367-375 (YLFPQLISV) HLA-A2 peptides, and to characterize the specific activities of XBP1 peptides-specific cytotoxic T lymphocytes (XBP1-CTL) against breast cancer, colon cancer, and pancreatic cancer cells.The XBP1-CTL had upregulated expression of critical T cell markers and displayed HLA-A2-restricted and antigen-specific activities against breast cancer, colon cancer and pancreatic cancer cells. XBP1-CTL were enriched withCD45RO+ memory CTL, which showed high expression of critical T cell markers (CD28, ICOS, CD69, CD40L), cell proliferation and antitumor activities as compared to CD45RO− non-memory CTL. The effector memory (EM: CD45RO+CCR7−) subset had the highest level of cell proliferation while the central memory (CM: CD45RO+CCR7+) subset demonstrated enhanced functional activities (CD107a degranulation, IFNγ/IL-2 production) upon recognition of the respective tumor cells. Furthermore, both the EM and CM XBP1-CTL subsets expressed high levels of Th1 transcription regulators Tbet and Eomes. The highest frequencies of IFNγ or granzyme B producing cells were detected within CM XBP1-CTL subset that were either Tbet+ or Eomes+ in responding to the tumor cells.These results demonstrate the immunotherapeutic potential of a cocktail of immunogenic HLA-A2 specific heteroclitic XBP1 US184-192 and heteroclictic XBP1 SP367-375 peptides to induce CD3+CD8+ CTL enriched for CM and EM cells with specific antitumor activities against a variety of solid tumors.
[Show abstract][Hide abstract] ABSTRACT: Carfilzomib, a recently FDA-approved proteasome inhibitor, has remarkable anti-myeloma (MM) activity. However, its effectiveness is limited by associated severe side-effects, short circulation half-life, and limited solubility. Here, we report the engineering of liposomal carfilzomib nanoparticles to overcome these problems and enhance the therapeutic efficacy of carfilzomib by increasing tumoral drug accumulation while decreasing systemic toxicity. In our design, carfilzomib was loaded into the bilayer of liposomes to yield stable and reproducible liposomal nanoparticles. Liposomal carfilzomib nanoparticles were efficiently taken up by MM cells, demonstrated proteasome inhibition, induced apoptosis, and exhibited enhanced cytotoxicity against MM cells. In vivo, liposomal carfilzomib demonstrated significant tumor growth inhibition and dramatically reduced overall systemic toxicity compared to free carfilzomib. Finally, liposomal carfilzomib demonstrated enhanced synergy in combination with doxorubicin. Taken together, this study establishes the successful synthesis of liposomal carfilzomib nanoparticles that demonstrate improved therapeutic index and the potential to improve patient outcome in MM.
No preview · Article · Oct 2014 · Journal of Controlled Release
[Show abstract][Hide abstract] ABSTRACT: New, next-generation targeted treatment strategies are required to improve outcomes in patients with multiple myeloma (MM). Monoclonal antibodies, cell signaling inhibitors, and selective therapies targeting the bone marrow microenvironment have demonstrated encouraging results with generally manageable toxicity in therapeutic trials of patients with relapsed and refractory disease, each critically informed by preclinical studies. A combination approach of these newer agents with immunomodulators and/or proteasome inhibitors as part of a treatment platform seems to improve the efficacy of anti-MM regimens, even in heavily pretreated patients. Future studies are required to better understand the complex mechanisms of drug resistance in MM.
No preview · Article · Oct 2014 · Hematology/Oncology Clinics of North America
[Show abstract][Hide abstract] ABSTRACT: Introduction: Multiple myeloma patients who are refractory to lenalidomide and bortezomib have a dismal prognosis. Pomalidomide is a new immunomodulatory agent approved for the treatment of relapsed and refractory multiple myeloma (RRMM) that is unique in that it demonstrates promising activity but appears to be associated with lower toxicity than thalidomide or lenalidomide.Areas covered: We review the mechanisms of action of pomalidomide, evaluate preclinical data, summarize the results of dose-finding Phase I studies and describe Phase II/III studies of this drug in combination with dexamethasone and other agents. Data presented were gathered from multiple sources, including articles from PubMed, published abstracts from the annual meetings of the American Society of Hematology and American Society of Clinical Oncology and websites such as http://clinicaltrials.gov/.Expert opinion: The regulatory approval of pomalidomide represents an important addition to a hematologist’s armamentarium for the treatment of RRMM. Pomalidomide is well tolerated and demonstrates a high level of anti-myeloma activity. Pomalidomide combined with dexamethasone should be considered as standard-of-care therapy for advanced RRMM following progression on both lenalidomide and bortezomib. Ongoing and future studies will characterize the activity of different combinations intended to improve treatment responses, and the potential role of pomalidomide as maintenance therapy.
No preview · Article · Sep 2014 · Expert Opinion on Orphan Drugs
[Show abstract][Hide abstract] ABSTRACT: Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma (MM), resistance to these drugs may arise via feedback activation loops. This concern is especially true for IGF-1 receptor (IGF-1R), because IGF-1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in MM using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S2481 as the readout for mTORC2/Akt activity in MM cells in the context of mTOR inhibition via AZD8055, or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in MM cells alone or in culture with BMSCs and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of MM cells. AZD8055 treatment however induced upregulation of IGF-1R phosphorylation in p-Akt S473 expressing MM cell lines. Furthermore, exposure of AZD8055-treated cells to IGF-1 induced p-Akt S473 and rescued MM cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF-1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF-1R blocking agents is a promising strategy in MM with potential IGF-1R/Akt signaling mediated survival.
Full-text · Article · Aug 2014 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] ABSTRACT: The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we therefore examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patients MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL-6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of CHOP, a fatal ER-stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM.
[Show abstract][Hide abstract] ABSTRACT: Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.