Michael F. Emmons

Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance

Imatinib mesylate is a potent, molecularly targeted therapy against the oncogenic tyrosine kinase BCR-ABL. Although imatinib mesylate has considerable efficacy against chronic myeloid leukemia (CML), advanced-stage CML patients frequently become refractory to this agent. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrices, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib mesylate sensitivity, we used an in vitro bone marrow stroma model. Our data show culturing K562 cells, in bone marrow stroma-derived conditioned medium (CM), is sufficient to cause resistance to BCR-ABL inhibitors. Drug resistance correlated with increased pTyrStat3, whereas no increases in pTyrStat5 was noted. Moreover, resistance was associated with increased levels of the Stat3 target genes Bcl-xl, Mcl-1, and survivin. Finally, reducing Stat3 levels with small interfering RNA sensitized K562 cells cultured in CM to imatinib mesylate-induced cell death. Importantly, Stat3 dependency was specific for cells grown in CM, as reducing Stat3 levels in regular growth conditions had no effect on imatinib mesylate sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib mesylate resistance and provides preclinical rationale for using Stat3-inhibitors to increase the efficacy of imatinib mesylate within the context of the bone marrow microenvironment. [Mol Cancer Ther 2008;7(10):3169–75]

Cell Adhesion to Fibronectin (CAM-DR) Influences Acquired Mitoxantrone Resistance in U937 Cells

Cell adhesion to fibronectin is known to confer a temporally related cell adhesion-mediated drug resistance (CAM-DR). However, it is unknown whether cell adhesion during drug selection influences the more permanent form of acquired drug resistance. To examine this question, we compared the acquisition of mitoxantrone resistance in U937 cells adhered to fibronectin versus cells selected in a traditional suspension culture. Our data show that acquired drug resistance levels of resistance to mitoxantrone are 2- to 3-fold greater for cells adhered to fibronectin compared with cells in suspension culture. We also compared mechanism(s) of resistance associated with drug selection in suspension versus fibronectin-adherent cultures. Drug resistance in both suspension and fibronectin-adhered cultures correlated with reduced drug-induced DNA damage and diminished topoisomerase II levels and activity; however, mechanisms regulating topoisomerase II levels differed depending on culture conditions. In suspension cultures, a reduction in topoisomerase IIbeta levels was detected at both RNA and protein levels. Furthermore, the decreased expression of topoisomerase IIbeta mRNA levels correlated with decreased expression of NF-YA. In contrast, in spite of no changes in NF-YA or topoisomerase IIbeta RNA expression, topoisomerase IIbeta protein levels were decreased in fibronectin-adherent, drug-resistant cells. In addition, topoisomerase IIalpha protein levels (but not RNA levels) were reduced in drug resistance cells selected on fibronectin; however, no change in topoisomerase IIalpha was observed in cells selected with mitoxantrone in suspension culture. Taken together, our results suggest that the development of drug resistance models must consider interactions with the microenvironment to identify clinically relevant targets and mechanisms associated with acquired drug resistance.

Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition.

Topoisomerase IIalpha (topo IIalpha) is exported from the nucleus of human myeloma cells by a CRM1-dependent mechanism at cellular densities similar to those found in patient bone marrow. When topo IIalpha is trafficked to the cytoplasm, it is not in contact with the DNA; thus, topo IIalpha inhibitors are unable to induce DNA-cleavable complexes and cell death. Using a CRM1 inhibitor or a CRM1-specific small interfering RNA (siRNA), we were able to block nuclear export of topo IIalpha as shown by immunofluorescence microscopy. Human myeloma cell lines and patient myeloma cells isolated from bone marrow were treated with a CRM1 inhibitor or CRM1-specific siRNA and exposed to doxorubicin or etoposide at high cell densities. CRM1-treated cell lines or myeloma patient cells were 4-fold more sensitive to topo II poisons as determined by an activated caspase assay. Normal cells were not significantly affected by CRM1-topo II inhibitor combination treatment. Cell death was correlated with increased DNA double-strand breaks as shown by the comet assay. Band depletion assays of CRM1 inhibitor-exposed myeloma cells showed increased topo IIalpha covalently bound to DNA. Topo IIalpha knockdown by a topo IIalpha-specific siRNA abrogated the CRM1-topo II therapy synergistic effect. These results suggest that blocking topo IIalpha nuclear export sensitizes myeloma cells to topo II inhibitors. This method of sensitizing myeloma cells suggests a new therapeutic approach to multiple myeloma.

HYD1-induced increase in reactive oxygen species leads to autophagy and necrotic cell death in multiple myeloma cells

HYD1 is a D–amino acid peptide that was previously shown to inhibit adhesion of prostate cancer cells to the extracellular matrix. In this study, we show that in addition to inhibiting adhesion of multiple myeloma (MM) cells to fibronectin, HYD1 induces cell death in MM cells as a single agent. HYD1-induced cell death was necrotic in nature as shown by: (a) decrease in mitochondrial membrane potential (Δψm), (b) loss of total cellular ATP, and (c) increase in reactive oxygen species (ROS) production. Moreover, HYD1 treatment does not result in apoptotic cell death because it did not trigger the activation of caspases or the release of apoptosis-inducing factor and endonuclease G from the mitochondria, nor did it induce double-stranded DNA breaks. HYD1 did initiate autophagy in cells; however, autophagy was found to be an adaptive response contributing to cell survival rather than the cause of cell death. We were further able to show that N-acetyl-L-cysteine, a thiol-containing free radical scavenger, partially protects MM cells from HYD1-induced death. Additionally, N-acetyl-L-cysteine blocked HYD1-induced as well as basal levels of autophagy, suggesting that ROS can potentially trigger both cell death and cell survival pathways. Taken together, our data describe an important role of ROS in HYD1-induced necrotic cell death in MM cells. [Mol Cancer Ther 2009;8(8):2441–51]

CRM1 Inhibition Sensitizes Drug Resistant Human Myeloma Cells to Topoisomerase II and Proteasome Inhibitors both In Vitro and Ex Vivo

Multiple myeloma (MM) remains an incurable disease despite improved treatments, including lenalidomide/pomalidomide and bortezomib/carfilzomib based therapies and high-dose chemotherapy with autologous stem cell rescue. New drug targets are needed to further improve treatment outcomes. Nuclear export of macromolecules is misregulated in many cancers, including in hematological malignancies such as MM. CRM1 (chromosome maintenance protein-1) is a ubiquitous protein that exports large proteins (>40 kDa) from the nucleus to the cytoplasm. We found that small-molecule Selective Inhibitors of Nuclear Export (SINE) prevent CRM1-mediated export of p53 and topoisomerase IIα (topo IIα). SINEs CRM1-inhibiting activity was verified by nuclear-cytoplasmic fractionation and immunocytochemical staining of the CRM1 cargoes p53 and topo IIα in MM cells. We found that SINE molecules reduced cell viability and induced apoptosis when used as both single agents in the sub-micromolar range and when combined with doxorubicin, bortezomib, or carfilzomib but not lenalidomide, melphalan, or dexamethasone. In addition, CRM1 inhibition sensitized MM cell lines and patient myeloma cells to doxorubicin, bortezomib, and carfilzomib but did not affect peripheral blood mononuclear or non-myeloma bone marrow mononuclear cells as shown by cell viability and apoptosis assay. Drug resistance induced by co-culture of myeloma cells with bone marrow stroma cells was circumvented by the addition of SINE molecules. These results support the continued development of SINE for patients with MM.

Acquisition of resistance toward HYD1 correlates with a reduction in cleaved α4 integrin expression and a compromised CAM-DR phenotype.

We recently reported that the β1 integrin antagonist, referred to as HYD1, induces necrotic cell death in myeloma cell lines as a single agent using in vitro and in vivo models. In this article, we sought to delineate the determinants of sensitivity and resistance toward HYD1-induced cell death. To this end, we developed an HYD1 isogenic resistant myeloma cell line by chronically exposing H929 myeloma cells to increasing concentrations of HYD1. Our data indicate that the acquisition of resistance toward HYD1 correlates with reduced levels of the cleaved α4 integrin subunit. Consistent with reduced VLA-4 (α4β1) expression, the resistant variant showed ablated functional binding to fibronectin, VCAM-1, and the bone marrow stroma cell line HS-5. The reduction in binding of the resistant cell line to HS-5 cells translated to a compromised cell adhesion-mediated drug resistant phenotype as shown by increased sensitivity to melphalan- and bortezomib-induced cell death in the bone marrow stroma coculture model of drug resistance. Importantly, we show that HYD1 is more potent in relapsed myeloma specimens than newly diagnosed patients, a finding that correlated with α4 integrin expression. Collectively, these data indicate that this novel d-amino acid peptide may represent a good candidate for pursuing clinical trials in relapsed myeloma and in particular patients with high levels of α4 integrin. Moreover, our data provide further rationale for continued preclinical development of HYD1 and analogues of HYD1 for the treatment of multiple myeloma and potentially other tumors that home and/or metastasize to the bone. Mol Cancer Ther; 10(12); 2257–66. ©2011 AACR.

The role of phenotypic plasticity in the escape of cancer cells from targeted therapy

Targeted therapy has proven to be beneficial at producing significant responses in patients with a wide variety of cancers. Despite initially impressive responses, most individuals ultimately fail these therapies and show signs of drug resistance. Very few patients are ever cured. Emerging evidence suggests that treatment of cancer cells with kinase inhibitors leads a minor population of cells to undergo a phenotypic switch to a more embryonic-like state. The adoption of this state, which is analogous to an epithelial-to-mesenchymal transition, is associated with drug resistance and increased tumor aggressiveness. In this commentary we will provide a comprehensive analysis of the mechanisms that underlie the embryonic reversion that occurs on targeted cancer therapy and will review potential novel therapeutic strategies designed to eradicate the escaping cells.

Expression of integrin alpha 10 is transcriptionally activated by pRb in mouse osteoblasts and is downregulated in multiple solid tumors

pRb is known as a classic cell cycle regulator whose inactivation is an important initiator of tumorigenesis. However, more recently, it has also been linked to tumor progression. This study defines a role for pRb as a suppressor of the progression to metastasis by upregulating integrin α10. Transcription of this integrin subunit is herein found to be pRb dependent in mouse osteoblasts. Classic pRb partners in cell cycle control, E2F1 and E2F3, do not repress transcription of integrin α10 and phosphorylation of pRb is not necessary for activation of the integrin α10 promoter. Promoter deletion revealed a pRb-responsive region between −108 bp to −55 bp upstream of the start of the site of transcription. pRb activation of transcription also leads to increased levels of integrin α10 protein and a greater concentration of the integrin α10 protein at the cell membrane of mouse osteoblasts. These higher levels of integrin α10 correspond to increased binding to collagen substrate. Consistent with our findings in mouse osteoblasts, we found that integrin α10 is significantly underexpressed in multiple solid tumors that have frequent inactivation of the pRb pathway. Bioinformatically, we identified data consistent with an ‘integrin switch’ that occurs in multiple solid tumors consisting of underexpression of integrins α7, α8, and α10 with concurrent overexpression of integrin β4. pRb promotes cell adhesion by inducing expression of integrins necessary for cell adhesion to a substrate. We propose that pRb loss in solid tumors exacerbates aggressiveness by debilitating cellular adhesion, which in turn facilitates tumor cell detachment and metastasis.

Emerging Strategies for Targeting Cell Adhesion in Multiple Myeloma

Multiple myeloma (MM) is an incurable hematological cancer involving proliferation of abnormal plasma cells that infiltrate the bone marrow (BM) and secrete monoclonal antibodies. The disease is clinically characterized by bone lesions, anemia, hypercalcemia, and renal failure. MM is presently treated with conventional therapies like melphalan, doxorubicin, and prednisone; or novel therapies like thalidomide, lenalidomide, and bortezomib; or with procedures like autologous stem cell transplantation. Unfortunately, these therapies fail to eliminate the minimal residual disease that remains persistent within the confines of the BM of MM patients. Mounting evidence indicates that components of the BM-including extracellular matrix, cytokines, chemokines, and growth factors-provide a sanctuary for subpopulations of MM. This co-dependent development of the disease in the context of the BM not only ensures the survival and growth of the plasma cells but contributes to de novo drug resistance. In addition, by fostering homing, angiogenesis, and osteolysis, this crosstalk plays a critical role in the progression of the disease. Not surprisingly then, over the past decade, several strategies have been developed to disrupt this communication between the plasma cells and the BM components including antibodies, peptides, and inhibitors of signaling pathways. Ultimately, the goal is to use these therapies in combination with the existing antimyeloma agents in order to further reduce or abolish minimal residual disease and improve patient outcomes.

MTI-101 (cyclized HYD1) binds a CD44 containing complex and induces necrotic cell death in multiple myeloma

Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma cell lines. Because of the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the linear peptide. These efforts led to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as a single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment similar to HYD1 induced reactive oxygen species, depleted ATP levels, and failed to activate caspase-3. Moreover, MTI-101 is cross-resistant in H929 cells selected for acquired resistance to HYD1. Here, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and liquid chromatography/tandem mass spectrometry analysis to identify binding partners of MTI-101. Using this approach, CD44 was identified as a predominant binding partner. Reducing the expression of CD44 was sufficient to induce cell death in multiple myeloma cell lines, indicating that multiple myeloma cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide. However, ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101–induced cell death. Mechanistically, we show that MTI-101–induced cell death occurs via a Rip1-, Rip3-, or Drp1-dependent and -independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma. Mol Cancer Ther; 12(11); 2446–58. ©2013 AACR.