Gertjan J. L. Kaspers

Molecular basis of bortezomib resistance: proteasome subunit β5 (PSMB5) gene mutation and overexpression of PSMB5 protein

The proteasome inhibitor bortezomib is a novel anticancer drug that has shown promise in the treatment of refractory multiple myeloma. However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the molecular basis of which remains elusive. Toward this end, we here developed high levels (45- to 129-fold) of acquired resistance to bortezomib in human myelomonocytic THP1 cells by exposure to stepwise increasing (2.5-200 nM) concentrations of bortezomib. Study of the molecular mechanism of bortezomib resistance in these cells revealed (1) an Ala49Thr mutation residing in a highly conserved bortezomib-binding pocket in the proteasome beta5-subunit (PSMB5) protein, (2) a dramatic overexpression (up to 60-fold) of PSMB5 protein but not of other proteasome subunits including PSMB6, PSMB7, and PSMA7, (3) high levels of cross-resistance to beta5 subunit-targeted cytotoxic peptides 4A6, MG132, MG262, and ALLN, but not to a broad spectrum of chemotherapeutic drugs, (4) no marked changes in chymotrypsin-like proteasome activity, and (5) restoration of bortezomib sensitivity in bortezomib-resistant cells by siRNA-mediated silencing of PSMB5 gene expression. Collectively, these findings establish a novel mechanism of bortezomib resistance associated with the selective overexpression of a mutant PSMB5 protein.

Aphidicolin decreases ex vivo resistance to cytosine arabinoside in childhood acute leukaemia

In order to assess the effect of the DNA polymerase inhibitor aphidicolin on resistance to cytosine arabinoside, blast cells from 15 children with ALL and 9 with AML were exposed to a range of concentrations of ara-C +/- aphidicolin. Cell survival was measured using the MTT assay. Aphidicolin significantly increased sensitivity to ara-C in blast cells from both ALL (p=0.001) and AML (p<0.01). The median fold increase (sensitisation ratio) for ALL was 3.4 (range 1.2-13.6) compared to 12.4-fold (range 6.0-148) for AML blasts (p=0.005). There was a striking relationship between increasing ara-C resistance and increasing effect of aphidicolin in AML (p<0.001) but not ALL (p>0.05). These remarkable results suggest that aphidicolin should be considered for future clinical trials as a modulator of ara-C resistance, particularly in AML.

Assessment of mercaptopurine (6MP) metabolites and 6MP metabolic key-enzymes in childhood acute lymphoblastic leukemia.

Pediatric acute lymphoblastic leukemia (ALL) is treated with combination chemotherapy including mercaptopurine (6MP) as an important component. Upon its uptake, 6MP undergoes a complex metabolism involving many enzymes and active products. The prognostic value of all the factors engaged in this pathway still remains unclear. This study attempted to determine which components of 6MP metabolism in leukemic blasts and red blood cells are important for 6MPs sensitivity and toxicity. In addition, changes in the enzymatic activities and metabolite levels during the treatment were analyzed. In a cohort (N = 236) of pediatric ALL patients enrolled in the Dutch ALL-9 protocol, we studied the enzymes inosine-5′-monophosphate dehydrogenase (IMPDH), thiopurine S-methyltransferase (TPMT), hypoxanthine guanine phosphoribosyl transferase (HGPRT), and purine nucleoside phosphorylase (PNP) as well as thioguanine nucleotides (TGN) and methylthioinosine nucleotides (meTINs). Activities of selected enzymes and levels of 6MP derivatives were measured at various time points during the course of therapy. The data obtained and the toxicity related parameters available for these patients were correlated with each other. We found several interesting relations, including high concentrations of two active forms of 6MP—TGN and meTIN—showing a trend toward association with better in vitro antileukemic effect of 6MP. High concentrations of TGN and elevated activity of HGPRT were found to be significantly associated with grade III/IV leucopenia. However, a lot of data of enzymatic activities and metabolite concentrations as well as clinical toxicity were missing, thereby limiting the number of assessed relations. Therefore, although a complex study of 6MP metabolism in ALL patients is feasible, it warrants more robust and strict data collection in order to be able to draw more reliable conclusions.

Abstract 4764: Transfer of regulatory protein networks via extracellular vesicles as a candidate mechanism of apoptosis-resistance in acute myeloid leukemia

Defects in apoptosis regulation are known to impact chemotherapy resistance and consequently refractoriness and relapse of acute myeloid leukemia (AML). We previously showed that apoptosis-resistant protein profile of AML blasts at diagnosis is associated with shorter disease-free survival. Specifically, by flow cytometry, we measured the expression of Bcl-2, Bcl-xL, Mcl-1 and Bax in leukemic cells and combined these parameters to define their anti-apoptosis index (AAI). Interestingly, the AAI of normal lymphocytes in the AML patients corresponded to the AAI of AML blasts obtained from the same patient, reaching values far outside the normal AAI range of lymphocytes. In addition, the AAI in both cell types displayed parallel changes during the course of therapy. This points to a role of microenvironment in regulation of apoptosis in bone marrow cells of AML patients. Therefore, the aim of the current study was to assess if apoptosis-resistant AML cells are able to regulate the AAI of apoptosis-sensitive cells by influencing the microenvironment, as well as to perform molecular dissection of microenvironment, to identify novel proteins that regulate apoptosis. First, we showed that apoptosis-resistant AML blasts (high AAI) release factors that modulate sensitive AML blasts (low AAI) to upregulate Bcl-2 and become apoptosis-resistant. In the majority of cases (10 out of 14), Bcl-2 expression was significantly increased in apoptosis-sensitive AML blasts upon contact culture with apoptosis-resistant AML blasts (1.7-fold; p=0.0067). To characterize the AML microenvironment, conditioned medium (18 hrs) from patient samples displaying either apoptosis-resistant (n=5) or apoptosis-sensitive profile (n=6) were collected. Using mass spectrometry-based proteomics, comparative analysis was performed on these secretomes. Strikingly, we found that the major functional protein clusters upregulated in secretomes of the apoptosis-resistant AML were involved in mRNA splicing, protein translation and chromatin remodeling/chromosome organization. We further compared protein profiles of the soluble secretome and the extracellular vesicle fraction of a high AAI patient to those of a low AAI patient. Proteomic analysis of these fractions of the conditioned medium showed that the functional protein networks found in the whole secretome are well-represented in extracellular vesicles that are enriched for exosome markers. Transfer of functional proteins between cells by extracellular vesicles is a well documented phenomenon. Therefore, it is conceivable that the regulatory protein networks detected in the vesicles excreted by AML blasts are involved in regulation of apoptosis-related proteins in recipient AML blasts and other cells residing in the bone marrow, thereby contributing to therapy resistance. Funded by STR and KiKa - Children cancer-free Citation Format: Anna Wojtuszkiewicz, Jacqueline Cloos, Floortje L. Kessler, Sander Piersma, Jako Knol, Gerrit Jansen, Yehuda G. Assaraf, Gertjan L. Kaspers, Sonja Zweegman, Gerrit J. Schuurhuis, Connie R. Jimenez. Transfer of regulatory protein networks via extracellular vesicles as a candidate mechanism of apoptosis-resistance in acute myeloid leukemia. [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 4764. doi:10.1158/1538-7445.AM2014-4764