Yiguo Hu

Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia

Targeting of cancer stem cells is believed to be essential for curative therapy of cancers, but supporting evidence is limited. Few selective target genes in cancer stem cells have been identified. Here we identify the arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regulator for leukemia stem cells (LSCs) in BCR-ABL–induced chronic myeloid leukemia (CML). In the absence of Alox5, BCR-ABL failed to induce CML in mice. This Alox5 deficiency caused impairment of the function of LSCs but not normal hematopoietic stem cells (HSCs) through affecting differentiation, cell division and survival of long-term LSCs (LT-LSCs), consequently causing a depletion of LSCs and a failure of CML development. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly by affecting LT-LSCs, and prolonged survival. These results demonstrate that a specific target gene can be found in cancer stem cells and its inhibition can completely inhibit the function of these stem cells.

Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma

Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P < .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.

Immunomodulatory effects of lenalidomide and pomalidomide on interaction of tumor and bone marrow accessory cells in multiple myeloma

The bone marrow (BM) microenvironment consists of extracellular-matrix and the cellular compartment including immune cells. Multiple myeloma (MM) cell and BM accessory cell interaction promotes MM survival via both cell-cell contact and cytokines. Immunomodulatory agents (IMiDs) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. Here we examined the in vitro effects of IMiDs on cytokine signaling triggered by interaction of effector cells with MM cells and BM stroma cells. IMiDs diminished interleukin-2, interferonγ, and IL-6 regulator suppressor of cytokine signaling (SOCS)1 expression in immune (CD4T, CD8T, natural-killer T, natural-killer) cells from both BM and PB of MM patients. In addition, coculture of MM cells with healthy PBMCs induced SOCS1 expression in effector cells; conversely, treatment with IMiDs down-regulated the SOCS1 expression. SOCS1 negatively regulates IL-6 signaling and is silenced by hypermethylation in MM cells. To define the mechanism of inhibitory-cytokine signaling in effector cells and MM cells, we next analyzed the interaction of immune cells with MM cells that were epigenetically modified to re-express SOCS1; IMiDs induced more potent CTL responses against SOCS1 re-expressing-MM cells than unmodified MM cells. These data therefore demonstrate that modulation of SOCS1 may enhance immune response and efficacy of IMiDs in MM.

Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor in the bone marrow microenvironment in multiple myeloma

Bruton tyrosine kinase (Btk) has a well-defined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis. Here we investigated effects of PCI-32765, an oral and selective Btk inhibitor, on osteoclastogenesis as well as on multiple myeloma (MM) growth within the BM microenvironment. PCI-32765 blocked RANKL/M-CSF-induced phosphorylation of Btk and downstream PLC-γ2 in OCs, resulting in diminished TRAP5b (ED50 = 17 nM) and bone resorption activity. PCI-32765 also inhibited secretion of multiple cytokines and chemokines from OC and BM stromal cell cultures from both normal donors (ED50 = 0.5 nM) and MM patients. It decreased SDF-1-induced migration of MM cells, and down-regulated MIP1-α/CCL3 in MM cells. It also blocked MM cell growth and survival triggered by IL-6 or coculture with BM stromal cells or OCs in vitro. Importantly, PCI-32765 treatment significantly inhibits in vivo MM cell growth (P < .03) and MM cell-induced osteolysis of implanted human bone chips in SCID mice. Moreover, PCI-32765 prevents in vitro colony formation by stem-like cells from MM patients. Together, these results delineate functional sequelae of Btk activation mediating osteolysis and growth of MM cells, supporting evaluation of PCI-32765 as a novel therapeutic in MM.

Inhibitory effects of omacetaxine on leukemic stem cells and BCR-ABL-induced chronic myeloid leukemia and acute lymphoblastic leukemia in mice

Omacetaxine mepesuccinate (formerly homoharringtonine) is a molecule with a mechanism of action that is different from tyrosine kinase inhibitors, and its activity in chronic myeloid leukemia (CML) seems to be independent of the BCR-ABL mutation status. Using BCR-ABL-expressing myelogenous and lymphoid cell lines and mouse models of CML and B-cell acute lymphoblastic leukemia (B-ALL) induced by wild-type BCR-ABL or T315I mutant-BCR-ABL, we evaluated the inhibitory effects of omacetaxine on CML and B-ALL. We showed that more than 90% of the leukemic stem cells were killed after treatment with omacetaxine in vitro. In contrast, less than 9 or 25% of the leukemic stem cells were killed after treating with imatinib or dasatinib, respectively. After 4 days of treatment of CML mice with omacetaxine, Gr-1+myeloid leukemia cells decreased in the peripheral blood of the treated CML mice. In the omacetaxine-treated B-ALL mice, only 0.8% of the B220+leukemia cells were found in peripheral blood, compared with 34% of the B220+leukemia cells in the placebo group. Treatment with omacetaxine decreased the number of leukemia stem cells and prolonged the survival of mice with BCR-ABL-induced CML or B-ALL.

Histone deacetylase 3 as a novel therapeutic target in multiple myeloma

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.

Dual Inhibition of Canonical and Noncanonical NF-κB Pathways Demonstrates Significant Antitumor Activities in Multiple Myeloma

Purpose: NF-κB transcription factor plays a key role in the pathogenesis of multiple myeloma in the context of the bone marrow microenvironment. Both canonical and noncanonical pathways contribute to total NF-κB activity. Recent studies have shown a critical role for the noncanonical pathway: selective inhibitors of the canonical pathway present a limited activity, mutations of the noncanonical pathway are frequent, and bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-κB activity. Experimental Design: Multiple myeloma cell lines, primary patient cells, and the human multiple myeloma xenograft murine model were used to examine the biologic impact of dual inhibition of both canonical and noncanonical NF-κB pathways. Results: We show that PBS-1086 induces potent cytotoxicity in multiple myeloma cells but not in peripheral blood mononuclear cells. PBS-1086 overcomes the proliferative and antiapoptotic effects of the bone marrow milieu, associated with inhibition of NF-κB activity. Moreover, PBS-1086 strongly enhances the cytotoxicity of bortezomib in bortezomib-resistant multiple myeloma cell lines and patient multiple myeloma cells. PBS-1086 also inhibits osteoclastogenesis through an inhibition of RANK ligand (RANKL)–induced NF-κB activation. Finally, in a xenograft model of human multiple myeloma in the bone marrow milieu, PBS-1086 shows significant in vivo anti–multiple myeloma activity and prolongs host survival, associated with apoptosis and inhibition of both NF-κB pathways in tumor cells. Conclusions: Our data show that PBS-1086 is a promising dual inhibitor of the canonical and noncanonical NF-κB pathways. Our preclinical study therefore provides the framework for clinical evaluation of PBS-1086 in combination with bortezomib for the treatment of multiple myeloma and related bone lesions. Clin Cancer Res; 18(17); 4669–81. ©2012 AACR.

Investigational agent MLN9708/2238 targets tumor-suppressor miR33b in MM cells

miRs play a critical role in tumor pathogenesis as either oncogenes or tumor-suppressor genes. However, the role of miRs and their regulation in response to proteasome inhibitors in multiple myeloma (MM) is unclear. In the current study, miR profiling in proteasome inhibitor MLN2238-treated MM.1S MM cells shows up-regulation of miR33b. Mechanistic studies indicate that the induction of miR33b is predominantly via transcriptional regulation. Examination of miR33b in patient MM cells showed a constitutively low expression. Overexpression of miR33b decreased MM cell viability, migration, colony formation, and increased apoptosis and sensitivity of MM cells to MLN2238 treatment. In addition, overexpression of miR33b or MLN2238 exposure negatively regulated oncogene PIM-1 and blocked PIM-1 wild-type, but not PIM-1 mutant, luciferase activity. Moreover, PIM-1 overexpression led to significant abrogation of miR33b- or MLN2238-induced cell death. SGI-1776, a biochemical inhibitor of PIM-1, triggered apoptosis in MM. Finally, overexpression of miR33b inhibited tumor growth and prolonged survival in both subcutaneous and disseminated human MM xenograft models. Our results show that miR33b is a tumor suppressor that plays a role during MLN2238-induced apoptotic signaling in MM cells, and these data provide the basis for novel therapeutic strategies targeting miR33b in MM.

Antimyeloma Activity of a Multitargeted Kinase Inhibitor, AT9283, via Potent Aurora Kinase and STAT3 Inhibition Either Alone or in Combination with Lenalidomide

Purpose: Aurora kinases, whose expression is linked to genetic instability and cellular proliferation, are being investigated as novel therapeutic targets in multiple myeloma (MM). In this study, we investigated the preclinical activity of a small-molecule multitargeted kinase inhibitor, AT9283, with potent activity against Aurora kinase A, Aurora kinase B, and Janus kinase 2/3. Experimental Design: We evaluated the in vitro antimyeloma activity of AT9283 alone and in combination with lenalidomide and the in vivo efficacy by using a xenograft mouse model of human MM. Results: Our data showed that AT9283 induced cell-growth inhibition and apoptosis in MM. Studying the apoptosis mechanism of AT9283 in MM, we observed features consistent with both Aurora kinase A and Aurora kinase B inhibition, such as increase of cells with polyploid DNA content, decrease in phospho-histone H3, and decrease in phospho-Aurora A. Importantly, AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells. Genetic depletion of STAT3, Aurora kinase A, or Aurora kinase B showed growth inhibition of MM cells, suggesting a role of AT9283-induced inhibition of these molecules in the underlying mechanism of MM cell death. In vivo studies showed decreased MM cell growth and prolonged survival in AT9283-treated mice compared with controls. Importantly, combination studies of AT9283 with lenalidomide showed significant synergistic cytotoxicity in MM cells, even in the presence of bone marrow stromal cells. Enhanced cytotoxicity was associated with increased inhibition of phosphorylated STAT3 and phosphorylated extracellular signal–regulated kinase. Conclusions: Demonstration of in vitro and in vivo anti-MM activity of AT9283 provides the rationale for the clinical evaluation of AT9283 as monotherapy and in combination therapy for treating patients with MM. Clin Cancer Res; 17(10); 3259–71. ©2011 AACR.