Denise Niewerth

Impaired bortezomib binding to mutant beta 5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells.

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the β5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZ-resistant cells revealed upregulation of mutant β5 subunit of the proteasome. These newly identified β5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the β5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant β5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the α7 subunit of the proteasome can overcome this drug resistance modality.

A review on allogeneic stem cell transplantation for newly diagnosed pediatric acute myeloid leukemia

Survival of pediatric acute myeloid leukemia (AML) has improved considerably over the past decades. Since 1985, allogeneic stem cell transplantation (allo-SCT) is widely recommended for patients who have a matched sibling donor. However, it remains controversial whether allo-SCT is superior to chemotherapy for children with newly diagnosed AML. This review summarizes phase 3 clinical trials that compared allo-SCT with chemotherapy (including autologous SCT) in pediatric AML, excluding studies that did not use the intention-to-treat analysis or correct for time-to-transplantation. Although allo-SCT might prevent more relapses than chemotherapy, the number needed for transplantation (with allo-SCT) to prevent one relapse is in the order of 10 patients. Moreover, overall survival is similar with both methods in most recent studies, apparently because of increased salvagability of a relapse when initial therapy concerned chemotherapy only, and because of a higher treatment-related mortality with allo-SCT. Because allo-SCT also gives more severe side effects and results more often in secondary malignancies than chemotherapy, we do not recommend allo-SCT in first remission for pediatric AML in general. Further research should focus on the possibility that subgroups might benefit from allo-SCT, aiming at further improvements in the prognosis of pediatric AML.

Molecular basis of resistance to proteasome inhibitors in hematological malignancies.

Over the past decade, the proteasome inhibitor bortezomib (Velcade) has not only gained a cornerstone position in the treatment of hematological malignancies, particularly multiple myeloma and mantle cell lymphoma, but also in experimental therapeutics of acute leukemia. However, the therapeutic efficacy of bortezomib is hampered by the emergence of acquired resistance, for which multifactorial mechanisms have been identified. This review summarizes the current status of the molecular mechanisms underlying resistance to proteasome inhibitors that emerged in preclinical therapeutic studies, and discusses these findings in the clinical perspective of novel therapeutic modalities of hematological malignancies. The specific topics that will be addressed in the current review include the recently established mechanisms of resistance to proteasome inhibitors: the role of constitutive and immunoproteasomes, mutations in proteasome subunits, unfolded protein response, XBP1 and MARCKS proteins, multidrug efflux transporters, aggresomes and autophagy, as well as the impact of pro-survival signaling pathways and bone marrow microenvironment. The growing knowledge of the determinants that confer bortezomib resistance and/or toxicity has provided the basis for the rational development of second generation proteasome inhibitors, some of which were recently approved or that are undergoing clinical evaluation as novel strategies to overcome bortezomib resistance as well as to enhance clinical therapeutic efficacy along with minimal side effects. Collectively, these combined approaches should enhance therapeutic efficacy and outcome in patients with hematological malignancies.

Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines

BackgroundDespite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, hence calling for novel strategies to overcome bortezomib-resistance. We previously showed that bortezomib-resistant human leukemia cell lines expressed significantly lower levels of immunoproteasome at the expense of constitutive proteasomes, which harbored point mutations in exon 2 of the PSMB5 gene encoding the β5 subunit. Here we investigated whether up-regulation of immunoproteasomes by exposure to interferon-γ restores sensitivity to bortezomib in myeloma and leukemia cell lines with acquired resistance to bortezomib.MethodsRPMI-8226 myeloma, THP1 monocytic/macrophage and CCRF-CEM (T) parental cells and sub lines with acquired resistance to bortezomib were exposed to Interferon-γ for 24-48 h where after the effects on proteasome subunit expression and activity were measured, next to sensitivity measurements to proteasome inhibitors bortezomib, carfilzomib, and the immunoproteasome selective inhibitor ONX 0914. At last, siRNA knockdown experiments of β5i and β1i were performed to identify the contribution of these subunits to sensitivity to proteasome inhibition. Statistical significance of the differences were determined using the Mann-Whitney U test.ResultsInterferon-γ exposure markedly increased immunoproteasome subunit mRNA to a significantly higher level in bortezomib-resistant cells (up to 30-fold, 10-fold, and 6-fold, in β1i, β5i, and β2i, respectively) than in parental cells. These increases were paralleled by elevated immunoproteasome protein levels and catalytic activity, as well as HLA class-I. Moreover, interferon-γ exposure reinforced sensitization of bortezomib-resistant tumor cells to bortezomib and carfilzomib, but most prominently to ONX 0914, as confirmed by cell growth inhibition studies, proteasome inhibitor-induced apoptosis, activation of PARP cleavage and accumulation of polyubiquitinated proteins. This sensitization was abrogated by siRNA silencing of β5i but not by β1i silencing, prior to pulse exposure to interferon-γ.ConclusionDownregulation of β5i subunit expression is a major determinant in acquisition of bortezomib-resistance and enhancement of its proteasomal assembly after induction by interferon-γ facilitates restoration of sensitivity in bortezomib-resistant leukemia cells towards bortezomib and next generation (immuno) proteasome inhibitors.

Higher ratio immune versus constitutive proteasome level as novel indicator of sensitivity of pediatric acute leukemia cells to proteasome inhibitors

The ex vivo sensitivity of pediatric leukemia cells to the proteasome inhibitor bortezomib was compared to 3 next generation proteasome inhibitors: the epoxyketone-based irreversible proteasome inhibitors carfilzomib, its orally bio-available analog ONX 0912, and the immunoproteasome inhibitor ONX 0914. LC50 values were determined by MTT cytotoxicity assays for 29 childhood acute lymphoblastic leukemia and 12 acute myeloid leukemia patient samples and correlated with protein expression levels of the constitutive proteasome subunits (β5, β1, β2) and their immunoproteasome counterparts (β5i, β1i, β2i). Acute lymphoblastic leukemia cells were up to 5.5-fold more sensitive to proteasome inhibitors than acute myeloid leukemia cells (P<0.001) and the combination of bortezomib and dexamethasone proved additive/synergistic in the majority of patient specimens. Although total proteasome levels in acute lymphoblastic leukemia and acute myeloid leukemia cells did not differ significantly, the ratio of immuno/constitutive proteasome was markedly higher in acute lymphoblastic leukemia cells over acute myeloid leukemia cells. In both acute lymphoblastic leukemia and acute myeloid leukemia, increased ratios of β5i/β5, β1i/β1 and β2i/β2 correlated with increased sensitivity to proteasome inhibitors. Together, differential expression levels of constitutive and immunoproteasomes in pediatric acute lymphoblastic leukemia and acute myeloid leukemia constitute an underlying mechanism of sensitivity to bortezomib and new generation proteasome inhibitors, which may further benefit from synergistic combination therapy with drugs including glucocorticoids.

Proteasome inhibitors in acute leukemia

Proteasome inhibition has been recognized as a novel treatment modality in hematologic malignancies. Initially, the reversible proteasome inhibitor bortezomib demonstrated efficacy in multiple myeloma (MM), which supported its approval for relapsed and refractory MM in 2003. Later on, carfilzomib, a next-generation irreversible proteasome inhibitor was approved by the US FDA in July 2012 for relapsed/refractory MM. Currently, several other proteasome inhibitors are undergoing preclinical and clinical evaluation. The successes of proteasome inhibitors in MM are now being translated to other hematologic malignancies, including acute leukemia. The first clinical studies with bortezomib in leukemia revealed promising clinical activity, particularly when combined with conventional chemotherapeutics. In this review the position of proteasome inhibitors in acute leukemia treatment is summarized and discussed. Special focus is also attributed to immunoproteasome inhibitors. As a future perspective, it is anticipated that proteasome inhibitors may prove to be of added value in therapeutic interventions for acute leukemia.

Antileukemic Activity and Mechanism of Drug Resistance to the Marine Salinispora tropica Proteasome Inhibitor Salinosporamide A (Marizomib)

Salinosporamide A (NPI-0052, marizomib) is a naturally occurring proteasome inhibitor derived from the marine actinobacterium Salinispora tropica, and represents a promising clinical agent in the treatment of hematologic malignancies. Recently, these actinobacteria were shown to harbor self-resistance properties to salinosporamide A by expressing redundant catalytically active mutants of the 20S proteasome β-subunit, reminiscent of PSMB5 mutations identified in cancer cells with acquired resistance to the founding proteasome inhibitor bortezomib (BTZ). Here, we assessed the growth inhibitory potential of salinosporamide A in human acute lymphocytic leukemia CCRF-CEM cells, and its 10-fold (CEM/BTZ7) and 123-fold (CEM/BTZ200) bortezomib-resistant sublines harboring PSMB5 mutations. Parental cells displayed sensitivity to salinosporamide A (IC50 = 5.1 nM), whereas their bortezomib-resistant sublines were 9- and 17-fold cross-resistant to salinosporamide A, respectively. Notably, combination experiments of salinosporamide A and bortezomib showed synergistic activity in CEM/BTZ200 cells. CEM cells gradually exposed to 20 nM salinosporamide A (CEM/S20) displayed stable 5-fold acquired resistance to salinosporamide A and were 3-fold cross-resistant to bortezomib. Consistent with the acquisition of a PSMB5 point mutation (M45V) in CEM/S20 cells, salinosporamide A displayed a markedly impaired capacity to inhibit β5-associated catalytic activity. Last, compared with parental CEM cells, CEM/S20 cells exhibited up to 2.5-fold upregulation of constitutive proteasome subunits, while retaining unaltered immunoproteasome subunit expression. In conclusion, salinosporamide A displayed potent antileukemic activity against bortezomib-resistant leukemia cells. β-Subunit point mutations as a common feature of acquired resistance to salinosporamide A and bortezomib in hematologic cells and S. tropica suggest an evolutionarily conserved mechanism of resistance to proteasome inhibitors.

Anti-leukemic activity and mechanisms underlying resistance to the novel immunoproteasome inhibitor PR-924

PR-924 is a novel prototypic immunoproteasome inhibitor bearing markedly enhanced specificity for the β5i immunoproteasome subunit, compared to the classical proteasome inhibitor bortezomib. Here, we assessed the growth inhibitory potential of PR-924 in three human hematologic malignancy cell lines (CCRF-CEM, THP1, and 8226) and their bortezomib-resistant sublines. Parental cells displayed equal sensitivity to PR-924 (IC₅₀: 1.5-2.8 μM), whereas their bortezomib-resistant tumor lines displayed a 10-12 fold cross-resistance to PR-924. However, PR-924 cross-resistance factors for bortezomib-resistant sublines were markedly lower compared to the resistance factors to bortezomib. Proteasome inhibition experiments confirmed that PR-924 specifically inhibited β5i activity, even far below concentrations that exerted anti-proliferative activity. We further determined whether PR-924 activity might be compromised by acquisition of drug resistance phenomena. Indeed, CEM cells rendered stepwise resistant to 20 μM PR-924 (CEM/PR20) displayed 13-fold PR-924-resistance and 10-fold cross-resistance to bortezomib. CEM/PR20 cells were devoid of mutations in the PSMB8 gene (encoding β5i), but acquired Met45Ile mutation in the PSMB5 gene (encoding constitutive β5), consistent with β5 mutations observed in bortezomib-resistant cells. Furthermore, compared to parental CEM cells, CEM/PR20 cells exhibited 2.5-fold upregulation of constitutive proteasome subunit expression, whereas immunoproteasome subunit expression was 2-fold decreased. In conclusion, PR-924 displayed potent anti-leukemic activity including toward bortezomib-resistant leukemia cells. Despite the specificity of PR-924 to the β5i immunoproteasome subunit, its anti-leukemic effect required concentrations that blocked both β5 and β5i subunits. This is underscored by the emergence of mutations in PSMB5 rather than in PSMB8.

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors

BackgroundIn clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence.MethodsHere we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC).ResultsCharacterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10–12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive β5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2–4 fold increase in the mRNA and protein levels of the constitutive β5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC.ConclusionsThese results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.

Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.