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Supplemental Methods, Supplementary Tables, Supplementary Figure Legends

Monocytes take up AF488-labeled daratumumab-CD38 complexes from PKH-26-stained UM9 cells.

Daratumumab-mediated CD38 reduction of MM cells in the presence of PBMCs as shown by Western blot analysis.

Loss of CD38 from the MM cell membrane is associated with reduced CDC and ADCC

Purpose: Daratumumab treatment results in a marked reduction of CD38 expression on multiple myeloma cells. The aim of this study was to investigate the clinical implications and the underlying mechanisms of daratumumab-mediated CD38 reduction.Experimental Design: We evaluated the effect of daratumumab alone or in combination with lenalidomide-dexamethasone, on CD38 levels of multiple myeloma cells and nontumor immune cells in the GEN501 study (daratumumab monotherapy) and the GEN503 study (daratumumab combined with lenalidomide-dexamethasone). In vitro assays were also performed.Results: In both trials, daratumumab reduced CD38 expression on multiple myeloma cells within hours after starting the first infusion, regardless of depth and duration of the response. In addition, CD38 expression on nontumor immune cells, including natural killer cells, T cells, B cells, and monocytes, was also reduced irrespective of alterations in their absolute numbers during therapy. In-depth analyses revealed that CD38 levels of multiple myeloma cells were only reduced in the presence of complement or effector cells, suggesting that the rapid elimination of CD38high multiple myeloma cells can contribute to CD38 reduction. In addition, we discovered that daratumumab–CD38 complexes and accompanying cell membrane were actively transferred from multiple myeloma cells to monocytes and granulocytes. This process of trogocytosis was also associated with reduced surface levels of some other membrane proteins, including CD49d, CD56, and CD138.Conclusions: Daratumumab rapidly reduced CD38 expression levels, at least in part, through trogocytosis. Importantly, all these effects also occurred in patients with deep and durable responses, thus excluding CD38 reduction alone as a mechanism of daratumumab resistance.The trials were registered at www.clinicaltrials.gov as NCT00574288 (GEN501) and NCT1615029 (GEN503). Clin Cancer Res; 23(24); 7498–511. ©2017 AACR.

Daratumumab-mediated CD38 reduction on NK cells is abrogated by depleting monocytes from PBMCs.

Vaccination guidelines for patients treated for hematological diseases are typically conservative. Given their high risk for severe COVID-19, it is important to identify those patients that benefit from vaccination. We prospectively quantified serum immunoglobulin G (IgG) antibodies to spike subunit 1 (S1) antigens during and after 2-dose mRNA-1273 (Spikevax/Moderna) vaccination in hematology patients. Obtaining S1 IgG ≥ 300 binding antibody units (BAUs)/mL was considered adequate as it represents the lower level of S1 IgG concentration obtained in healthy individuals, and it correlates with potent virus neutralization. Selected patients (n = 723) were severely immunocompromised owing to their disease or treatment thereof. Nevertheless, >50% of patients obtained S1 IgG ≥ 300 BAUs/mL after 2-dose mRNA-1273. All patients with sickle cell disease or chronic myeloid leukemia obtained adequate antibody concentrations. Around 70% of patients with chronic graft-versus-host disease (cGVHD), multiple myeloma, or untreated chronic lymphocytic leukemia (CLL) obtained S1 IgG ≥ 300 BAUs/mL. Ruxolitinib or hypomethylating therapy but not high-dose chemotherapy blunted responses in myeloid malignancies. Responses in patients with lymphoma, patients with CLL on ibrutinib, and chimeric antigen receptor T-cell recipients were low. The minimal time interval after autologous hematopoietic cell transplantation (HCT) to reach adequate concentrations was

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity, however, IXA more potently inhibited β1i subunit than BTZ (70% vs 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells, mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

Contains fulltext : 220824.pdf (Publisher’s version ) (Open Access) Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic myeloid progenitors. Aberrantly activated tyrosine kinases (TK) are involved in leukemogenesis and are associated with poor treatment outcome. Kinase inhibitor (KI) treatment has shown promise in improving patient outcome in AML. However, inhibitor selection for patients is suboptimal. In a preclinical effort to address KI selection, we analyzed a panel of 16 AML cell lines using phosphotyrosine (pY) enrichment-based, label-free phosphoproteomics. The Integrative Inferred Kinase Activity (INKA) algorithm was used to identify hyperphosphorylated, active kinases as candidate for KI treatment, and efficacy of selected KIs was tested. Heterogeneous signaling was observed with between 241 and 2764 phosphopeptides detected per cell line. Of 4853 identified phosphopeptides with 4229 phosphosites, 4459 phosphopeptides (4430 pY) were linked to 3605 class I sites (3525 pY). INKA analysis in single cell lines successfully pinpointed driver kinases (PDGFRA, JAK2, KIT and FLT3) corresponding with activating mutations present in these cell lines. Furthermore, potential receptor tyrosine kinase (RTK) drivers, undetected by standard molecular analyses, were identified in four cell lines (FGFR1 in KG-1 and KG-1a, PDGFRA in Kasumi-3, and FLT3 in MM6). These cell lines proved highly sensitive to specific KIs. Six AML cell lines without a clear RTK driver showed evidence of MAPK1/3 activation, indicative of the presence of activating upstream RAS mutations. Importantly, FLT3 phosphorylation was demonstrated in two clinical AML samples with a FLT3 internal tandem duplication (ITD) mutation. Our data show the potential of pY-phosphoproteomics and INKA analysis to provide insight in AML TK signaling and identify hyperactive kinases as potential targets for treatment in AML cell lines. These results warrant future investigation of clinical samples to further our understanding of TK phosphorylation in relation to clinical response in the individual patient.

In non‐small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR‐inhibitor erlotinib and the cMET‐inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live‐cell microscopy. We observed additive effects in EBC‐1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR‐inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live‐cell microscopy with a pH‐sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib–erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions., In non‐small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR‐inhibitor erlotinib and the cMET‐inhibitor crizotinib. Both genetic characteristics and lysosomal function seem to contribute to the synergistic interaction between crizotinib and erlotinib in the HCC827GR5 cell line, and more research is warranted to determine the exact mechanisms linking these cellular properties.

Purpose: Daratumumab treatment results in a marked reduction of CD38 expression on multiple myeloma cells. The aim of this study was to investigate the clinical implications and the underlying mechanisms of daratumumab-mediated CD38 reduction. Experimental Design: We evaluated the effect of daratumumab alone or in combination with lenalidomide-dexamethasone, on CD38 levels of multiple myeloma cells and nontumor immune cells in the GEN501 study (daratumumab monotherapy) and the GEN503 study (daratumumab combined with lenalidomide-dexamethasone). In vitro assays were also performed. Results: In both trials, daratumumab reduced CD38 expression on multiple myeloma cells within hours after starting the first infusion, regardless of depth and duration of the response. In addition, CD38 expression on nontumor immune cells, including natural killer cells, T cells, B cells, and monocytes, was also reduced irrespective of alterations in their absolute numbers during therapy. In-depth analyses revealed that CD38 levels of multiple myeloma cells were only reduced in the presence of complement or effector cells, suggesting that the rapid elimination of CD38high multiple myeloma cells can contribute to CD38 reduction. In addition, we discovered that daratumumab–CD38 complexes and accompanying cell membrane were actively transferred from multiple myeloma cells to monocytes and granulocytes. This process of trogocytosis was also associated with reduced surface levels of some other membrane proteins, including CD49d, CD56, and CD138. Conclusions: Daratumumab rapidly reduced CD38 expression levels, at least in part, through trogocytosis. Importantly, all these effects also occurred in patients with deep and durable responses, thus excluding CD38 reduction alone as a mechanism of daratumumab resistance. The trials were registered at www.clinicaltrials.gov as NCT00574288 (GEN501) and NCT1615029 (GEN503). Clin Cancer Res; 23(24); 7498–511. ©2017 AACR.

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p = 0.0067 and p = 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p = 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.

The clinical efficacy of proteasome inhibitors in the treatment of multiple myeloma has encouraged application of proteasome inhibitor containing therapeutic interventions in (pediatric) acute leukemia. Here, we summarize the positioning of bortezomib, as first-generation proteasome inhibitor, and second-generation proteasome inhibitors in leukemia treatment from a preclinical and clinical perspective. Potential markers for proteasome inhibitor sensitivity and/or resistance emerging from leukemia cell line models and clinical sample studies will be discussed focusing on the role of immunoproteasome and constitutive proteasome (subunit) expression, PSMB5 mutations, and alternative mechanisms of overcoming proteolytic stress.

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.

// Niels E. Franke 1 , Gertjan L. Kaspers 1 , Yehuda G. Assaraf 2 , Johan van Meerloo 1, 3 , Denise Niewerth 1 , Floortje L. Kessler 3 , Pino J. Poddighe 4 , Jeroen Kole 9 , Serge J. Smeets 5 , Bauke Ylstra 5 , Chonglei Bi 6, 11 , Wee Joo Chng 6 , Terzah M. Horton 7 , Rene X. Menezes 8 , Renee J.P. Musters 9 , Sonja Zweegman 3 , Gerrit Jansen 10, * , Jacqueline Cloos 1, 3, * 1 Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands 2 The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa, Israel 3 Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands 4 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands 5 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 6 Department of Experimental Therapeutics, Cancer Science Institute of Singapore, National University of Singapore, Singapore 7 Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX, USA 8 Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands 9 Department of Physiology, VU University, Amsterdam, The Netherlands 10 Department of Rheumatology, Amsterdam Rheumatology and immunology Center, VU University Medical Center, Amsterdam, The Netherlands 11 Current address: BGI-Shenzhen, Shenzhen, China * Joint senior authors Correspondence to: Jacqueline Cloos, email: j.cloos@vumc.nl Keywords: bortezomib, resistance, proteasome, MARCKS, leukemia Received: October 19, 2015 Accepted: July 26, 2016 Published: August 17, 2016 ABSTRACT 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.

Background Drug combinations of the proteasome inhibitor bortezomib with cytotoxic chemotherapy are currently evaluated in phase 2 and 3 trials for the treatment of paediatric acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL). Methods We investigated whether expression ratios of immunoproteasome to constitutive proteasome in leukaemic cells correlated with response to bortezomib-containing re-induction chemotherapy in patients with relapsed and refractory acute leukaemia, enrolled in two Children’s Oncology Group phase 2 trials of bortezomib for ALL (COG-AALL07P1) and AML (COG-AAML07P1). Expression of proteasome subunits was examined in 72 patient samples (ALL n = 60, AML n = 12) obtained before start of therapy. Statistical significance between groups was determined by Mann-Whitney U test. Results Ratios of immunoproteasome to constitutive proteasome subunit expression were significantly higher in pre-B ALL cells than in AML cells for both β5i/β5 and β1i/β1 subunits (p = 0.004 and p

Purpose Despite the encouraging results of bortezomib (BTZ) in hematologic malignancies to date, resistance to BTZ may be a limiting factor to its efficacy. Hence, parameters that may identify responsiveness to BTZ-containing therapy will be of clinical interest. Recently, we reported that higher ratios of immunoproteasome over constitutive proteasome protein expression in pediatric ALL and AML leukemia cells at diagnosis were an accountable factor for ex vivo sensitivity to proteasome inhibitors (Niewerth et al, Haematologica 2013). Here we explored whether this parameter was associated with response to BTZ in first relapsed and refractory pediatric acute leukemia patients treated in phase II clinical trials of BTZ combined with re-induction chemotherapy for pediatric ALL (COG-AALL07P1) and pediatric AML (COG-AAML07P1). Methods Protein expression levels of constitutive- β5 and β1, and immunoproteasome subunits β5i and β1i were determined by Western blot analysis in 61 acute leukemia patient samples (ALL n=47, AML n=14) obtained before BTZ-containing reinduction therapy. In addition, β5 and β5i proteasome catalytic activities were measured in 14 ALL and 13 AML samples prior to treatment. Lastly, NF-ĸB activity was determined by p65 ELISA in nuclear extracts of PBMCs before and 24h after BTZ treatment. Results In pre-treatment samples, expression ratios of both β5i/β5 and β1i/β1 were significantly higher in ALL cells than in AML cells (P=0.049 and P=0.002, respectively). Ratios of both β5i/β5 and β1i/β1 were significantly higher in patients that reached complete remission (CR; n=39) compared to patients that did not reach CR (n=22) (P=0.009 for β5i/β5, P=0.025 for β1i/β1). Moreover, increased ratios of β5i/β5 catalytic activity were observed in pre-treatment ALL+AML samples that reached CR compared to those that did not reach CR (P=0.078). Proteasome activity ratios correlated significantly with proteasome expression ratios (R=0.55 P=0.005). Notably, NF-ĸB activity was similar in both groups and was suppressed after BTZ treatment, being most pronounced in the pre-B ALL patients that achieved CR (average decrease: 47% p=0.05). Conclusion These results suggest that a higher ratio of immuno/constitutive proteasome in pretreatment ALL and AML cells is an accountable factor for the clinical response to BTZ. These results warrant further investigation to establish a biomarker that can be used for selecting relapsed pediatric acute leukemia patients eligible for BTZ-containing reinduction treatment. This study was sponsored by KiKa (Children Cancer-free-GJLK), Millennium pharmaceuticals (TMH), and NIH-K23-CA113775 (TMH) Citation Format: Denise Niewerth, Gertjan J.L. Kaspers, Gerrit Jansen, Johan van Meerloo, Sonja Zweegman, Gaye Jenkins, James A. Whitlock, Stephen P. Hunger, Xiaomin Lu, Jacqueline Cloos, Terzah M. Horton. Ratios of immunoproteasome over constitutive proteasome expression are an indicator for sensitivity to bortezomib-containing reinduction chemotherapy in pediatric relapsed ALL and AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-169. doi:10.1158/1538-7445.AM2014-LB-169

Background Despite 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. Methods RPMI-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. Results Interferon-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. Conclusion Downregulation 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.

Background In 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. Methods Here 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). Results Characterization 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. Conclusions These 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.

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

The proteasome inhibitor bortezomib, registered for Multiple Myeloma treatment, is currently explored for activity in solid tumors including non-small cell lung cancer (NSCLC). Here we studied the proteasome-based mechanisms underlying intrinsic and acquired bortezomib resistance in NSCLC cells. Various NSCLC cell lines displayed differential intrinsic sensitivities to bortezomib. High basal chymotrypsin- and caspase-like proteasome activities correlated with bortezomib resistance in these cells. Next, via stepwise selection, acquired bortezomib resistant cells were obtained with 8-70-fold increased resistance. Cross-resistance was found to proteasome inhibitors specifically targeting beta-subunits, but not to the novel alpha-subunit-specific proteasome inhibitor (5AHQ). Consistently, bortezomib-resistant cells required higher bortezomib concentrations to induce G2/M arrest and apoptosis. Interestingly, bortezomib concentration-dependent caspase cleavage, Mcl-1 and NOXA accumulation remained intact in resistant H460 and SW1573 cells, while A549 resistant cells displayed different expression profiles suggesting additional and more protein specific adaptations. Furthermore, bortezomib-resistant cells exhibited increased levels of both constitutive and immuno-beta-subunits. Sequence analysis of the bortezomib-binding pocket in the beta 5-subunit revealed Ala49Thr, Met45Val and Cys52Phe substitutions that were not previously described in solid tumors. Bortezomib-resistant cells displayed reduced catalytic proteasome activities and required higher bortezomib concentrations to achieve comparable inhibition of proteasome activity. Taken together, these findings establish that high basal levels of proteasome activity correlate with intrinsic bortezomib resistance. Furthermore, acquired bortezomib resistance in NSCLC is associated with proteasome subunit overexpression and emergence of mutant beta 5-subunits that likely compromise bortezomib binding, alpha-Subunit-specific proteasome inhibitors, however, can efficiently bypass this resistance modality. (C) 2011 Elsevier Inc. All rights reserved.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity. Since for most cell populations the total mitochondrial activity is related to the number of viable cells, this assay is broadly used to measure the in vitro cytotoxic effects of drugs on cell lines or primary patient cells. In this chapter the protocol of the assay is described including important considerations relevant for each step of the assay as well as its limitations and possible applications.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity. Since for most cell populations the total mitochondrial activity is related to the number of viable cells, this assay is broadly used to measure the in vitro cytotoxic effects of drugs on cell lines or primary patient cells. In this chapter the protocol of the assay is described including important considerations relevant for each step of the assay as well as its limitations and possible applications.

A subgroup of head and neck squamous cell carcinomas (HNSCCs) contains high-risk human papillomavirus-type 16 (HPV16). The viral E6 and E7 oncoproteins inactivate the p53 and pRb proteins, respectively. We examined the causative effect of HPV16 E6 and E7 expression on the immortalization of normal oral keratinocytes (OKCs) and compared the resulting phenotype with alternative ways of p53- and pRb-pathway abrogation frequently found in HNSCCs without HPV. Primary OKCs were conditionally immortalized with temperature-sensitive SV40 large T-antigen and human telomerase, allowing these cells to return to their senescent primary state after temperature shift. HPV16 E6 and E7 were introduced to overcome senescence, determined with population doubling (PD) as read-out. For comparison, we downregulated p53 and p16 by short hairpin RNA genes and expressed mutant p53R(175)H and cyclinD1. Expression of HPV16 E6 caused an extended life span similar to expression of mutant p53R(175)H or p53 knockdown. Expression of mutant p53R(175)H seemed to cause additional activation of the hypoxia and WNT signaling pathways. HPV16 E7 expression had no direct effect on lifespan, similar to p16 knockdown or cyclinD1 expression. In combination with HPV16 E6 or other functional inactivations of p53, abrogation of the pRb-pathway by either HPV16 E7 or other manipulations caused an immortal phenotype. Our data show the causative role of HPV16 E6/E7 in early squamous carcinogenesis. Activity of each gene could be mimicked by other genetic events frequently found in HNSCC without HPV. This data provides the experimental proof of causal association of HPV in HNSCC carcinogenesis and further support the crucial role of the p53- and pRb-pathways.

Proteasome inhibition with bortezomib (BTZ) is an effective treatment for hematological malignancies and an emerging treatment strategy for acute leukemia. However, the side-effects of BTZ and emergence of BTZ-resistance call for alternative therapeutic approaches. The immunoproteasome may serve as a candidate since its expression is higher than constitutive proteasome expression in cells of hematological malignancies, including acute lymphocytic leukemia (ALL). To this end, the new immunoproteasome inhibitor PR-924 may elicit anti-leukemic activity, since it bears greater specificity for the β5i subunit of the immunoproteasome compared to other proteasome inhibitors including BTZ and carfilzomib. The aim of the current study was to examine the proteasome inhibition capacity and cell growth inhibitory impact of PR-924 in acute leukemia cells and sublines with acquired resistance to BTZ. We further determined whether PR-924 itself would be prone to resistance development and if so, to explore the underlying molecular basis. To assess the anti-leukemic activity of PR-924, its cytotoxicity was determined in two human cell lines of hematological origin: the T-ALL cell line CCRF-CEM and the acute myeloid leukemia cell line THP1, and their 150-fold BTZ-resistant sublines CEM/BTZ200, and THP1/BTZ200, respectively. Parental CEM and THP1 cells displayed similar sensitivity to PR-924 (IC50 CEM: 1.8 µM ± 0.4, THP1: 1.5 µM ± 0.2), whereas their BTZ-resistant lines displayed a moderate 10-12 fold cross-resistance to PR-924 (IC50 CEM/BTZ200: 21.1 µM ± 0.5, THP1/BTZ200: 15.6 µM ± 1.6). Flow cytometric analysis revealed that PR-924-induced cell death was mediated by induction of apoptosis. Moreover, PR-924 exposure resulted in a (up to 50%) reduction of cell surface expression of HLA Class I. To determine whether PR-924 activity relies on specific inhibition of β5i, proteasome activity inhibition experiments were performed over a range of 10 nM – 10 µM PR-924. Notably, in parental cells, β5i activity was already inhibited by 20% at 10 nM PR-924 and > 90% inhibition was achieved at 100 nM PR-924. At higher PR-924 concentrations of 1-10 µM, inhibition of both β5 and β1i activities was observed, thus indicating that PR-924 blocks the β5i activity far below concentrations that exert anti-proliferative activity (1.5 µM), consistent with earlier studies of PR-924 (Parlati et al. Blood, 2009). Next, acquired resistance to PR-924 was provoked in CEM and THP1 cells by step-wise increasing PR-924 concentrations in cell culture, starting at the IC50 concentrations. Following this process, CEM cells resistant to 20 µM PR-924 (CEM/PR20) and THP1 cells resistant to 12 mM PR-924 (THP1/PR12) exhibited IC50 values of 22.1 µM PR-924 (resistance factor 13) and 14.3 µM PR-924 (resistance factor 10), respectively. In addition, these PR-924 resistant cells displayed 10-fold cross-resistance to BTZ. To explore mechanisms of PR-924 resistance, we first sequenced exon 2/3 of the PSMB8 gene (encoding β5i), a similar functioning coding region that was previously reported to harbor PSMB5 mutations in BTZ-resistant leukemia cells (Franke et al. Leukemia, 2012). However, no mutations were found in PSMB8 exon 2/3. Remarkably, we did identify mutations in exon 2 of the PSMB5 gene (encoding the S1 pocket of β5) in both CEM/PR20 (Met45Ile) and of THP1/PR12 (Ala49Thr); these amino acid substitutions are similar to those observed in BTZ-resistant sublines. Finally, we characterized proteasome subunit expression by ProCISE analysis in the PR-924 resistant cell lines. This method is a subunit-specific active-site ELISA assay, which utilizes a purified proteasome standard curve to calculate ng of subunit per µg total protein. Expression of constitutive proteasome subunits in CEM/PR20 and THP1/PR8 cells were upregulated up to 2.5-fold, compared to their parental counterparts, whereas immunoproteasome subunit expression was moderately decreased (up to 2-fold) in CEM/PR20 and increased in THP1/PR8 (up to 1.6-fold). In conclusion, PR-924 displayed significant anti-leukemic activity. Although there was cross-resistance to BTZ, PR-924 retained activity in BTZ-resistant leukemia cells. Despite PR-924 specificity to the β5i subunit of the proteasome, its anti-leukemic effect requires concentrations that block both β5 and β5i subunits. This notion is underscored by emergence of acquired mutations in PSMB5 rather than in PSMB8. Disclosures: Kirk: Onyx Pharmaceuticals: Employment, Equity Ownership. Anderl:Onyx: Employment.

1346 Acquired resistance to the proteasome inhibitor (PI) bortezomib (BTZ) is an emerging factor limiting its efficacy in the treatment of hematologic malignancies. The clinical impact of acquired resistance has been shown in Multiple Myeloma (MM) patients who were re-treated with BTZ. Although BTZ-retreatment was found to be effective, the response rate as well as the duration of response were less as compared to initial treatment, indicating the development of BTZ-resistance in a subgroup of patients. In line with that, we previously found increased expression of constitutive proteasome (cP) subunit s5 harbouring a mutation in the BTZ-binding pocket and a decreased expression of non-mutated immunoproteasome subunits in BTZ-resistant cell lines of hematologic malignancies (Franke and Niewerth et al, Leukemia 2012). We here explore whether upregulation of immunoproteasome (iP) expression could restore sensitivity in BTZ-resistant leukemia cells towards BTZ and two epxoyketone-based irreversible PIs; carfilzomib (CFZ) and the s5i-targeted ONX 0914. BTZ-resistant cell lines were of multiple myeloma (8226), T-cell (CEM) and myelomonocytic (THP1) origin and displayed resistance towards cell growth inhibition in the presence of 7–200 nM BTZ. Induction of iP in wild type (WT) and BTZ-resistant 8226, CCRF-CEM and THP1 cells was achieved by exposure to 100U/ml Interferon- γ (IFN-γ) for 6–72 h. IFN-γ transiently increased (maximum between 24–48 hours) mRNA levels of β5i, β1i, and β2i up to 8-fold, 30-fold and 4-fold, respectively. These findings were corroborated at the β5i, β1i and β2i protein expression level using Western blot analysis. Following IFN-γ exposure, chymotrypsin-like proteasome activity increased up to 2.5-fold compared to unstimulated controls, trypsin-like activity increased up to 1.5-fold, whereas caspase-like activity was slightly decreased. Consistent with increased proteasome activity, there was also an increased expression of cell surface HLA Class I molecules. The impact of IFN-γ induced upregulation of iPs on the sensitivity to the PI BTZ, CFZ, and ONX 0914, defined by the decrease in IC50, is summarized in [Table 1][1]. 8226/BTZ100 cells became 4-fold more sensitive towards BTZ after IFN-γ exposure, whereas THP1/BTZ200 and CEM/BTZ200 cells displayed nearly 2-fold increased sensitivity. For CFZ, a modest level of sensitization was observed in all cell lines with high level BTZ resistance. Interestingly, for the immunoproteasome inhibitor ONX 0914, IC50 values were markedly decreased (7-fold for 8226/BTZ100 and 3-fold for THP1/BTZ200 and CEM/BTZ200 cells). Additionally, in 8226 cells with low levels of BTZ resistance (8226/BTZ7), IFN-γ restored parental cell sensitivity to ONX 0914. Restoration of PI sensitivity after IFN-γ exposure was further confirmed by activation of PARP cleavage and accumulation of ubiquitinated proteins, pointing to restoration of BTZ activity under proteasome inhibition and consequent induction of apoptosis. Finally, to provide evidence that upregulation of β5i and or β1i by IFN-γ was responsible for the observed sensitization, siRNA downregulation of β5i and β1i was applied prior to exposure to IFN-γ. Under these conditions, mRNA levels and proteasome activity of β5i remained suppressed, even after exposure to IFN-γ. Moreover, after β5i silencing, PI sensitization and apoptosis were attenuated. Silencing of β1i expression had no effect on PI-sensitization. In conclusion, down-regulation of β5i subunit expression is a major determinant of BTZ-resistance and increasing its proteasomal assembly after IFN-γ exposure facilitates restoration of sensitivity in BTZ-resistant leukemia cells towards cP inhibitors and in particular iP inhibitors. | Cell lines | BTZ | BTZ + IFNy | SF | ONX 0914 | ONX 0914 + IFNy | SF | CFZ | CFZ + IFNy | SF | |:-----------:| ---- | ---------- | --- | -------- | --------------- | --- | --- | ---------- | --- | | 8226/wt | 2.6 | 1.8 | 1.4 | 54 | 46 | 1.5 | 0.4 | 0.4 | 1 | | 8226/BTZ7 | 13.5 | 5.8 | 2.3 | 99 | 47 | 2.1 | 0.9 | 0.8 | 1.1 | | 8226/BTZ100 | 208 | 57 | 3.6 | 1837 | 249 | 7.4 | 28 | 13 | 2.2 | | CEM/wt | 4.1 | 3.9 | 1.1 | 75 | 65 | 1.2 | 0.4 | 0.3 | 1.3 | | CEM/BTZ200 | 416 | 223 | 1.9 | 1763 | 566 | 3.1 | 42 | 26 | 1.6 | | THP1/wt | 6.2 | 5.1 | 1.2 | 52 | 19 | 2.7 | 0.9 | 1.3 | 0.7 | | THP1/BTZ200 | 641 | 347 | 1.8 | 4236 | 1376 | 3.1 | 49 | 34 | 1.4 | Table 1. IC50 values of PIs ± IFN-γ pre-incubation (48 hr) of wild type and BTZ-resistant hematologic cell lines 50% inhibitory concentration compared to untreated controls (IC50 nM) as determined in a 4 days growth inhibition assay (MTT). Results depicted are means of at least 3 separate experiments. SF: sensitization factor: IC50 control/IC50 with IFN-g. Disclosures: No relevant conflicts of interest to declare. [1]: #T1

Abstract 1513 Good response to glucocorticoids (GC) has favorable prognostic value for the survival of children with acute lymphoblastic leukemia (ALL). Hence, GC-resistant and relapsed ALL patients may benefit from GC-sensitization strategies. For this purpose, the reversible proteasome inhibitor (PI) Bortezomib (BTZ) is currently being evaluated in clinical trials in combination with Dexamethasone (DEX) and other drugs. Despite the encouraging results of BTZ in several hematological malignancies, emergence of resistance to BTZ may be a limiting factor to its efficacy. Therefore, the aim of our study was to examine the differential sensitivity of pediatric leukemia cells to BTZ and DEX, as compared to second generation PIs designed to overcome BTZ resistance. These include the epoxyketone-based irreversibly binding PIs Carfilzomib (CFZ), its orally bioavailable analog ONX 0912, and the immunoproteasome inhibitor ONX 0914. The drug concentration required for 50% cell death (LC50) was determined in pediatric patient samples (29 ALL and 12 AML) after 4 days drug exposure using the MTT cytotoxicity assay. Furthermore, the sensitivity to PIs was correlated with protein expression levels of the constitutive proteasome subunits beta5, beta1 and beta2, and the (immuno) proteasome subunits beta5i and beta1i. ALL cells were significantly more sensitive for BTZ than AML cells (median LC50: 6.0 nM vs 14.2 nM, respectively, p=0.002), and also markedly more sensitive to Dex (median LC50: 23.0 nM vs. >600 nM, p60012600.0164–>6000.000Subunit expressionRatio*Ratio* beta5280.760.00–30.0106.02.2–23.90.080 beta5i2762.58.5–3661055.010.6–3400.714 beta1282.40.00–28.11011.70.92–26.10.029 beta1i2835.15.42–1061017.77.2–49.50.032 beta2284.80.38–23.41020.47.4–39.10.000 beta2iN.D.N.D.N.D.N.D.N.D.N.D.N.D.N.D.: Not Determined. * Ratio proteasome subunit / β-actin based on loading of 15 ug total protein (Western blot analysis) For ALL, LC50 concentrations for CFZ and ONX 0912 were significantly correlated (r=0.449, p=0.019). Interestingly, for AML, a significant correlation was observed between BTZ and CFZ LC50 concentrations (r=0.900, p=0.001), suggestive for overlapping activities. Expression of constitutive proteasome subunits is higher in AML cells than ALL cells. Within ALL samples, constitutive proteasome subunit expression did not correlate with LC50 concentrations for each of the PIs. Within AML patients, however, beta 5 expression significantly correlated with BTZ LC50 (r=0.980, p In conclusion, ALL cells were more sensitive to PIs than AML, which may be due lower constitutive proteasome unit expression. Pediatric leukemia cells display marked sensitivity to BTZ and second generation PIs, but lack cross-resistance between BTZ and several second generation PIs. Together, for second generation PIs, these data may hold promise for circumvention of BTZ resistance and further exploration of efficacy assessments in combination with other drugs, in particular GCs. Disclosures: No relevant conflicts of interest to declare.

The proteasome inhibitor bortezomib (Velcade®) is registered for the treatment of multiple myeloma, but has limited activity in solid tumors such as non-small cell lung cancer (NSCLC). Bortezomib predominantly inhibits proteasome subunit β5 and mutations in this subunit were recently associated with bortezomib resistance in leukemic cell line models. Herein we studied the molecular mechanisms underlying intrinsic and acquired bortezomib resistance in NSCLC cells. H460, A549 and SW1573 NSCLC cells demonstrated differential intrinsic sensitivities towards bortezomib with IC50 values of 12.6, 9.0 and 2.1 nM, respectively. Basal proteasome activities in these cell lines measured by an intact cell proteasome activity assay revealed that high basal levels of chymotrypsin- and caspase-like proteasome activities strongly correlate with intrinsic bortezomib resistance (R2=0.99, P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2011-746

The proteasome inhibitor Bortezomib (BTZ, Velcade®) specifically inhibits the catalytic beta 5 subunit of the proteasome. The introduction of BTZ has shown promising results in the treatment of Multiple Myeloma (MM), Non-Hodgkin lymphoma and leukemia. Despite these encouraging results, clinical trials in MM also revealed that a significant proportion of patients acquired resistance to BTZ mono-therapy. We have developed a BTZ-resistant cell line model by chronic exposure to stepwise increasing concentrations of BTZ, including the AML THP-1 cell-line (Oerlemans & Franke et al, Blood 2008), the T-ALL CCRF-CEM-C7 cell-line and the MM RPMI-8226 cell line (Franke et al, Blood 2009 vol 114(22), abstr 940). Cells were initially selected for growth at 7 nM BTZ to acquire low levels of BTZ resistance (2–3 fold higher IC50 concentrations) and subsequently challenged to concentrations of BTZ up to 500 nM to provoke higher resistance levels. Abilities to resist a BTZ concentration of 100 nM could be achieved relatively fast in the CEM cell line (within 16 weeks), intermediate in the THP-1 cell line (within 22 weeks) and relatively slowly in the 8226 cell line (within 60 weeks). Subsequent sequencing of the PSMB5 gene, encoding the beta 5 proteasome subunit, revealed a series of mutations in individual BTZ-resistant subclones, all resulting in amino-acid changes residing within the highly conserved BTZ binding pocket. The relatively fast induction of mutations provoked the question whether there might already exist a subclone within the cell line that harbours the mutation. To distinguish between the outgrowth of a pre-existing resistant subclone and the occurrence of de novo mutations, we generated new BTZ resistant CEM and THP-1 cells (CEM-BR2 and THP-1-BR2). Interestingly, the new CEM-BR2 cells had a different nucleotide change (G322A resulting in a Ala49Thr substitution) from the original BTZ resistant CEM-BR1 (C323T and G332T resulting in Ala49Val and Cys52Phe amino acid substitutions, respectively). Of note, this new CEM-BR2 mutation represented the same mutation as seen in the original BTZ resistant THP-1-BR1. In addition, the new THP-1-BR2 cells also showed a different mutation (A309G) in the PSMB5 gene, introducing a Met45Val amino-acid substitution) compared to the original BTZ resistant THP-1 cells that showed a Met45Iso substitution. These results indicate that the mutations are acquired during BTZ exposure and are mainly induced in specific hot-spots (dominantly Ala49) within the PSMB5 gene. To explore whether mutation-induced resistance could be bypassed, a new proteasome inhibitor, 5-amino-8-hydroxyquinole (5AHQ), acting as a non-competitive inhibitor of the non-catalytic alpha-7 subunit of the proteasome (Li et al. ASH 2008) was studied in our model system. Strikingly, all BTZ-resistant selectants retained full sensitivity towards 5AHQ (IC50: 4–7 μM, measured in a 4-day MTT cytotoxicity assay) as compared to parental cells. To determine whether mutation induction also occurs in patients treated with BTZ, screening for their presence in clinical samples of BTZ refractory patients is warranted. The notion that 5AHQ can overcome BTZ resistance related to single and multiple mutations in the PSMB5 gene, supports further research in this drug and its analogues. This study is supported by VUmc - Stichting Translational Research (STR) and The Netherlands Organization for Health Research and Development (ZonMw). Citation Information: Clin Cancer Res 2010;16(7 Suppl):A27

Abstract 940 The proteasome inhibitor Bortezomib (BTZ, Velcade®) specifically inhibits the catalytic beta 5 subunit of the proteasome, thereby interfering in the degradation of proteins involved in cell-cycling, NF-kB activation, apoptosis induction and micro-environmental interactions. The introduction of BTZ has shown promising results in the treatment of Multiple Myeloma (MM), Non-Hodgkin lymphoma and leukemia. Despite these encouraging results, clinical trials in MM also revealed that a significant proportion of patients acquired resistance to BTZ mono-therapy. Previously we reported the development of three different BTZ-resistant cell line models, including the AML THP-1 cell-line (Oerlemans & Franke et al, Blood 2008), the T-ALL CCRF-CEM-C7 cell-line and the MM RPMI-8226 cell line (Franke et al, ASH 2008) after chronic exposure to stepwise increasing concentrations of BTZ. The aim of our current study was to further elucidate the molecular basis of BTZ resistance in these cell lines and investigate whether the resistant phenotype could be overcome by the second generation proteasome inhibitor 5-amino-8-hydroxyquinole (5AHQ), acting as a non-competitive inhibitor of the non-catalytic alpha-7 subunit of the proteasome (Li et al. ASH 2008). When added to intact cells, cell extracts, or isolated rabbit reticulocyte proteasomes (gift of A Navon, Weizmann Institute of Science, Israel), 5AHQ inhibited the chymotrypsin-like enzymatic activity of the proteasome with IC50's of 6.9 ± 0.05 uM, 4.2 ± 0.6 uM, and 2.1 ± 0.18 μM, respectively. Cells were initially selected for growth at 7 nM BTZ to acquire low levels of BTZ resistance (2-3 fold higher IC50 concentrations) and subsequently challenged to higher concentrations of BTZ (up to 500 nM) to provoke higher resistance levels. Sequencing of the PSMB5 gene, encoding for the beta 5 proteasome subunit, revealed a series of mutations in individual BTZ-resistant subclones. An overview of the mutation site in the PSMB5 gene, the amino acid position at which substitutions take place at the beta 5 subunit protein level, and the functional implication of each mutation are depicted in the table below. Subsequently, we determined whether THP-1, CEM and 8226 cells with low and high levels of BTZ resistance would be sensitive towards the new proteasome inhibitor 5AHQ. Strikingly, all BTZ-resistant selectants retained full sensitivity towards 5AHQ (IC50: 4-7 μM, measured in a 4-day MTT cytotoxicity assay) as compared to parental cells. Together, these data indicate that in vitro selection of low and high levels of BTZ resistance in 3 hematological cell lines is accompanied by the introduction of multiple mutations of amino acids, all of which have key positions in BTZ binding within the active site of the proteasome beta 5 subunit. Notably, Ala49 mutations were independently observed in all 3 cell lines selected for BTZ concentrations > 100 nM. Knowledge of specific mutations in the PSMB5 gene that can confer BTZ resistance allows purposive screening for these types of mutations in clinical samples of BTZ refractory patients. In this respect, the notion that 5AHQ can bypass BTZ resistance related to single or multiple mutations in the PSMB5 gene, supports further preclinical development and clinical application of this drug. This study is supported by VUmc - Stichting Translational Research (STR) and The Netherlands Organization for Health Research and Development (ZonMw), The Leukemia and Lymphoma Society and the Ontario Institute for Cancer Research through funding from the Ministry of Research and Innovation, Province of Ontario. Disclosures: No relevant conflicts of interest to declare.