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]
Topoisomerase IIA (topo IIA) 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 IIA is trafficked to the cytoplasm, it is not in contact with the DNA; thus, topo IIA 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 IIA 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 IIA covalently bound to DNA. Topo IIA knockdown by a topo IIA-specific siRNA abrogated the CRM1-topo II therapy synergistic effect. These results suggest that blocking topo IIA nuclear export sensitizes myeloma cells to topo II inhibitors. This method of sensitizing myeloma cells suggests a new therapeutic approach to multiple myeloma. [Cancer Res 2009;69(17):6899-905]
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). SINE's 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.
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 fibronectinadherent cultures. Drug resistance in both suspension and fibronectin-adhered cultures correlated with reduced druginduced 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 IIB levels was detected at both RNA and protein levels. Furthermore, the decreased expression of topoisomerase IIB mRNA levels correlated with decreased expression of NF-YA. In contrast, in spite of no changes in NF-YA or topoisomerase IIB RNA expression, topoisomerase IIB protein levels were decreased in fibronectin-adherent, drug-resistant cells. In addition, topoisomerase IIA protein levels (but not RNA levels) were reduced in drug resistance cells selected on fibronectin; however, no change in topoisomerase IIA 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. (Cancer Res 2006; 66(4): 2338-45)
Purpose: The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. This study has used multiomics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance. Experimental Design: Mass spectrometry-based proteomics and RNA-Seq were used to identify the signaling pathways involved in the escape of uveal melanoma cells from MEK inhibitor therapy. Mechanistic studies were performed to evaluate the escape pathways identified, and the efficacy of the MEK-HDAC inhibitor combination was demonstrated in multiple in vivo models of uveal melanoma. Results: We identified a number of putative escape pathways that were upregulated following MEK inhibition, including the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. MEK inhibition was also associated with increased GPCR expression, particularly the endothelin B receptor, and this contributed to therapeutic escape through ET-3-mediated YAP signaling. A screen of 289 clinical grade compounds identified HDAC inhibitors as potential candidates that suppressed the adaptive YAP and AKT signaling that followed MEK inhibition. In vivo, the MEK-HDAC inhibitor combination outperformed either agent alone, leading to a long-term decrease of tumor growth in both subcutaneous and liver metastasis models and the suppression of adaptive PI3K/AKT and YAP signaling. Conclusions: Together, our studies have identified GPCRmediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma.
Increased HDAC8 activity deacetylates c-JUN, leading to increased EGFR signaling and BRAF inhibitor resistance Low HDAC8 activity RTK c-Jun High HDAC8 activity BRAFi c-Jun Ras RAF ERK Cell death Survival and invasion BRAFi Ras RAF ERK RTK (EGFR) ac ac ac TRE ac ac TRE p TR HDAC8 Melanoma cells have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanomas to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia, and UV irradiation leads to an increase in histone deacetylase 8 (HDAC8) activity and the adoption of a drugresistant phenotype. Mass spectrometry-based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling. Introduction of HDAC8 into drug-na€ ve melanoma cells conveyed resistance both in vitro and in vivo. HDAC8mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase activation, leading to MAPK signaling. Although HDACs function at the histone level, they also regulate nonhistone substrates, and introduction of HDAC8 decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine 273 increased transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both nonselective and HDAC8-specific inhibitors enhancing the durability of BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors limit the adaptation of melanoma cells to multiple stresses including BRAF-MEK inhibition. Significance: This study provides evidence that HDAC8 drives transcriptional plasticity in melanoma cells in response to a range of stresses through direct deacetylation of c-Jun.
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 report we sought to delineate the determinants of sensitivity and resistance towards HYD1 induced cell death. To this end, we developed a HYD1 isogenic resistant myeloma cell line by chronically exposing H929 meyloma cells to increasing concentrations of HYD1. Our data indicate that the acquisition of resistance towards 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 CAM-DR phenotype as demonstrated by increased sensitivity to melphalan and bortezomib induced cell death in the bone marrow stroma co-culture model of drug resistance. Importantly, we show that HYD1 is more potent in relapsed myeloma specimens compared to newly diagnosed patients, a finding which correlated with α4 integrin expression. Collectively, these data indicate that this novel D-amino acid peptide may represent a good candidate for pursing clinical trials in relapsed myeloma and in particular patients with high levels of α4 integrin. Moreover, our data provide further rationale for continued pre-clinical development of HYD1 and analogs of HYD1 for the treatment of multiple myeloma and potentially other tumors which home and/or metastasize to the bone.
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.
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