Proteasome inhibitors such as Bortezomib represent an established type of targeted treatment for several types of hematological malignancies, including multiple myeloma, Waldenstrom’s macroglobulinemia, and mantle cell lymphoma, based on the cancer cell’s susceptibility to impairment of the proteasome-ubiquitin system. However, a major problem limiting their efficacy is the emergence of resistance. Their application to solid tumors is currently being studied, while simultaneously, a wide spectrum of hematological cancers, such as Myelodysplastic Syndromes show minimal or no response to Bortezomib treatment. In this study, we utilize the prostate cancer cell line DU-145 to establish a model of Bortezomib resistance, studying the underlying mechanisms. Evaluating the resulting resistant cell line, we observed restoration of proteasome chymotrypsin-like activity, regardless of drug presence, an induction of pro-survival pathways, and the substitution of the Ubiquitin-Proteasome System role in proteostasis by induction of autophagy. Finally, an estimation of the oxidative condition of the cells indicated that the resistant clones reduce the generation of reactive oxygen species induced by Bortezomib to levels even lower than those induced in non-resistant cells. Our findings highlight the role of autophagy and oxidative stress regulation in Bortezomib resistance and elucidate key proteins of signaling pathways as potential pharmaceutical targets, which could increase the efficiency of proteasome-targeting therapies, thus expanding the group of molecular targets for neoplastic disorders.

The rapid emergence of resistance limits the application of proteasome inhibitors against solid tumors, despite their effectiveness in the treatment of hematological malignancies. Resistant phenotypes are complex and multifaceted, and, thus, the mechanisms involved have not been adequately described. In this study, a Bortezomib-resistant prostate cancer cell line is created by using the PC-3 cell as a prostate carcinoma model of high metastatic potential. The main biochemical differences and adaptations exhibited by the resistant cells revolve around apoptosis evasion, autophagy induction (functioning as a ubiquitin-proteasome system substitute), expression of epithelial-to-mesenchymal transition markers, and increased aggressiveness. Broad-spectrum signaling pathway analyses also reveal an upregulation and activation of Nf-κB, STAT3, cJun, and Elk1 transcription factors in the resistant cells. Additionally, intracellular reactive oxygen species assays reveal a downregulation in resistant cells, which is theorized to be a consequence of metabolic changes, increased autophagic flux, and antioxidative enzyme action. These findings expand our understanding of proteasome inhibitor resistance and highlight key kinases and transcription factors as novel potential therapeutic targets. Effective inhibition of resistance-specific pathways could re-sensitize the cells to proteasome inhibitors, thus surpassing current therapeutic limitations.

Proteasome inhibitors (PIs) constitute the most common type of induction treatment for multiple myeloma. Interactions between the proteasome, autophagy, and reactive oxygen species (ROS) have been shown in the past, thus emphasizing the need for a better understanding of the underlying pathophysiology. For this study, bone marrow mononuclear cells from 110 myeloma patients were collected at different disease stages. PSMB5 and LC3I/II protein levels were determined using Western blot, proteasome proteolytic activity (PPA) with spectrofluorometry, and ROS with flow cytometry. PSMB5 accumulation was found to diminish after PI treatment (p-value = 0.014), and the same pattern was observed in PPA (p-value < 0.001). Conversely, LC3II protein levels were elevated at both remission and relapse compared to baseline levels (p-value = 0.041). Patients with a baseline PSMB5 accumulation lower than 1.06 units had longer disease-free survival compared to those with values above 1.06 units (12.0 ± 6.7 vs. 36 ± 12.1 months; p-value < 0.001). Median ROS levels in plasma cells were significantly higher at relapse compared to both baseline and remission levels (p-value < 0.001), implying poor prognosis. Overall, post-treatment PSMB5 reduction could indicate a shift from proteasomal to autophagic degradation as a main proteostatic mechanism, thus explaining resistance. The elevated oxidative stress in PI-treated patients could possibly serve as an additional compensatory mechanism.

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and an elevated risk of transformation to acute myeloid leukemia (AML). Available disease-modifying treatment approaches are limited. The ineffectiveness of proteasome inhibitors (PIs) in MDS patients is currently investigated, although it is unclear whether they rapidly develop resistance to PIs or whether proteasome proteolytic activity (PPA) is constitutively lower in the hematopoietic cells of these patients, thus limiting treatment effectiveness. We investigated 20 patients with MDS, categorized according to the International Prognostic Scoring System (IPSS) into a lower- or a higher-risk group. Peripheral blood mononuclear cells, bone marrow mononuclear cells, and cluster of differentiation 34-positive (CD34+) cells were isolated and assessed for the chymotrypsin-like activity of the proteasome and β5 subunit accumulation. Additionally, intracellular reactive oxygen species (ROS) generation was screened. The lower-risk patient group (n=10) exhibited significantly lower proteasome activity (p<0.001) compared to both the higher-risk group (n=10) and healthy subjects (n=10). Furthermore, the lower-risk group had elevated oxidative stress levels (p<0.0001) and reduced β5 subunit expression (p=0.0286). Both parameters were shown to be associated with transfusion dependency, since transfusion-dependent patients (n=5 in each subgroup) had decreased proteasome activity and simultaneously exhibited higher ROS levels. Our results indicate that reduced β5 expression might potentially explain PIs' ineffectiveness in lower-risk MDS, elucidating the importance of the risk group in the selection of the proper treatment algorithm.

The effectiveness of proteasome inhibitors against solid tumors is limited as the emergence of resistance is rapid. Although many mechanisms have been proposed and verified, no definite answer has been given, highlighting the complexity of the resistant phenotype. In this study, a Bortezomib-resistant prostate cancer cell line is created, and a broad-spectrum signaling pathway analysis is performed to identify differences and adaptations the resistant cells exhibit. Our findings highlight the upregulation and activation of Nf-κB, STAT3, cJun, and Elk1 transcription factors in the resistant cells and the subsequent evasion of apoptosis and induction of autophagy, which is constantly activated and substitutes the role of the ubiquitin-proteasome system (UPS). Additionally, assessment of the intracellular reactive oxygen species in resistant cells confirms their downregulation, which is theorized to be a consequence of metabolic changes, increased autophagic flux, and antioxidative enzyme action. The results of this study highlight the potential therapeutic targeting of key kinases and transcription factors, participating in the main signaling pathways and gene regulation of Bortezomib-resistant cells, that could re-sensitize the cells to proteasome inhibitors, thus surpassing the current limitations.

Introduction: Azacitidine (AZA) is a hypomethylating agent that at low doses acts by inhibiting DNA methyltranferase activity. AZA is approved and widely used for the treatment of MDS patients and patients with AML not candidate for intensive chemotherapy. Unfortunately, even after an initial response, almost all patients relapse and so far -with the exception of a few clinical parameters and genetic mutations weakly correlated with favorable AZA response- the exact mechanisms underlying primary AZA resistance remain largely unknown. On the other hand, over the last years accumulated data suggest that hypoxia, an important regulatory factor of both, physiological and malignant, hematopoiesis, is also involved in MDS pathogenesis (Hayashi et al., 2018), while high Hif-1α levels in MDS have been previously correlated with poor overall survival and disease progression (Tong et al., 2012). Moreover, our group recently investigated the association between Hif-1α and response to AZA therapy and found that AZA-responders present with higher Hif-1α mRNA expression compared to non-responders/stable disease patients, while logistic regression analysis showed that Hif-1α mRNA expression is an independent predictor of response to AZA therapy (unpublished data). Aims: The current study focused on investigating the mechanisms underlying the observed association of Hif-1α over-expression with response to AZA-therapy, by examining the methyltransferase activity and mitochondrial dysfunction due to inactivation of complex II, which is reported to lead to increased Hif-1α expression. Methods: A total of 54 patients with a median age of 76 (52-89) years, and 10, age matched, healthy donors participated in the study. According to WHO 2016, 41 patients were classified as MDS (10 as MDS-EB-1, 24 as MDS-EB-2 and 7 as MDS-MLD) and 13 as AML. All patients received AZA treatment at the dose of 75mg/m² x7 days SC. BM-derived mononuclear cells were isolated before treatment using the Ficoll-paque method, followed by RNA extraction using TRIzol reagent, and cDNA preparation using Superscript II reverse transcriptase. Hypoxia-inducible factor 1-alpha (Hif-1α), succinate dehydrogenase complex subunit D (DSHd) and DNA methyltrasferase beta (DNMT3b) expression were estimated by real time PCR TaqMan gene expression assays, using the appropriate primers and probes. Relative gene expression was calculated by comparative threshold cycle (2^-ΔΔCt) method and normalized based on β-actin expression. Non-parametric tests were used for the statistical analysis of the results. Results: Out of the 54 examined patients, 28 responded to azacitidine treatment (R), (including CR, PR and HI), 9 failed to respond (NR), and 17 achieved stable disease status 9 (SD). NR and SD patients were considered as one group (NR/SD) in all analyses. Using Rt-PCR we found that the 2^-ΔΔCt ratio of Hif-1α/β-Actin median expression for control samples was 1.18 (95% CI: 0.617-1.687), for AZA-responders 1.59 (95% CI: 1.029-3.18), while for NR/SD patients 0.754 (95% CI: 0.640-0.840), with a statistical significance between R and NR/SD patients (Mann-Whitney test, p=0.003). Moreover, the 2^-ΔΔCt ratio of SDHd/β-Actin median expression for control samples was 1.2 (95% CI: 0.360-1.954), for R patients 0.81 (95% CI: 0.294-1.401), and for NR/SD patients 0.73 (95% CI: 0.542-0.793). Finally, for DNMT3b, the 2^-ΔΔCt median expression ratio in control samples was 0.75 (95% CI: 0.637-1.526), for R patients 2.188 (95% CI: 1.547-3.630), while for NR/SD patients 1.338 (95% CI: 0.824-2.250). Conclusions: Our data suggest that both AZA-R and NR/SD patients present with low levels of SDHd mRNA, compared to control, in line with previous reports in MDS. For AZA-responders, this could be related to the observed Hif-1α mRNA over-expression, since the SDH inactivation (decreased Complex II activity) is known to cause HIF stabilization (Frezza et al., 2011; Selak et al., 2005). Nevertheless, NR/SD patients also appear with decreased SDHd activity, despite the observed low Hif-1α expression. Therefore, in those patients, Hif-1α- related AZA-therapy response seems to be independent from mitochondrial dysfunction and possibly relies on other hypoxia regulatory mechanisms. Moreover, our data suggest that AZA-responders appear with an increased DNMT3b expression compared to both control and NR/SD patients, which could also explain their better response to therapy.