The current study describes two ways to overcome acquired resistance to BTZ in JY human B cells: (a) anti-CD20/rituximab-mediated CDC, and (b) next generation epoxyketone-based irreversible PIs.
B cell depleting therapies utilizing the anti-CD20/rituximab antibody approach, as well as the small molecule approach with the proteasome inhibitor BTZ, have demonstrated clinical efficacy in the treatment of lymphoproliferative disorders
[1, 10, 23, 48]. In the treatment of autoimmune diseases, rituximab also now has an accepted place
[3–5, 43], but BTZ or other PIs still await clinical evaluation. Ideally, based on their non-overlapping mechanisms of action, rituximab and bortezomib could potentially replace each other when development of resistance to one or the other occurred. In this context, it has been reported that rituximab-resistant B cells retain sensitivity for BTZ, but the reverse has not been demonstrated. In view of the need for chronic treatments in autoimmune diseases, there are concerns about the emergence of resistance
, thus highlighting the need to unravel underlying mechanisms of resistance, in order to design strategies to overcome acquired resistance. To address these issues, we provoked acquired resistance to BTZ in a human B cell line model and investigated its impact on rituximab sensitivity and next-generation PIs designed to overcome BTZ-resistance.
Here we mimicked the onset of acquired BTZ resistance in human B cells using human JY lymphoblastic cells as benign autoimmune disorder in vitro model, by a classical approach of cell exposure to stepwise increasing concentrations of BTZ (5 through 100 nM), representing clinically-achievable plasma levels
[10, 23]. Characterization of the BTZ-resistant JY cells (10–12 fold compared to WT cells) revealed the following main features: 1) acquisition of a point mutation in the PSMB5 gene (encoding for the constitutive β5 proteasome subunit) along with overexpression of β5 subunit protein, 2) retention of sensitivity to epoxyketone-based PIs including carfilzomib, 3) an altered cytokine environment indicated by a marked reduction in the secretion of the inflammatory cytokine TNF-α, and 4) immunophenotypically, a significantly reduced expression of the B cell surface marker CD19 and a concomitant increase in CD20 levels.
BTZ-resistance associated with a single point mutation in PSMB5 has been demonstrated by us and others for leukemia cell lines of lymphoid and myeloid origins, as well as solid tumor cell lines
[38–41, 49]. These mutations introduced amino acid alterations that clustered in critical positions (Thr21, Met45, Ala49 and Cys52) in the BTZ binding pocket within the β5 subunit
. Based on data from the present study, it is revealing that beyond hematological malignant cells, PSMB5 mutations can also be identified in BTZ-resistant JY lymphoblastic cells. It is noteworthy that the mutation in PSMB5 in JY/BTZ cells is heterozygous, indicating that cells still retained an intact WT allele. This may underlie a relatively lower level of BTZ resistance observed in JY/BTZ cells as compared to the substantially higher resistance levels (> 100-fold) noted in leukemic cells harboring homozygous mutations in PSMB5
[38, 41]. Notwithstanding this fact, expression levels of mutant β5 subunits were upregulated in JY/BTZ cells, likely as a compensatory mechanism to sustain basal proteasome activity. The Met45Ile substitution identified in BTZ-resistant JY cells consistently resided in the highly-conserved substrate/inhibitor-binding domain of the β5 subunit. Met45 is well recognized to be essential for proteasome-substrate interactions, as well as for binding of the leucine boronic acid moiety (P1 site) of BTZ
[29, 32, 50]. In this context, it may be anticipated that replacement of the non-polar, spatially flexible Met45 for another slightly more non-polar, bulkier, and more rigid amino acid as Ile, would affect optimal BTZ binding, thereby resulting in the conferring of drug resistance. From this perspective, it is intriguing to note that the Ala49 and Met45 mutations in the β5 subunit were also identified in marine bacteria as a self-protection mechanism against the natural PI, salinosporamide A, that they produce
A major finding in the current study is that JY/BTZ cells harboring the Met45Ile mutation in the β5 subunit retained full sensitivity to carfilzomib, an epoxyketone-based PI. Previous studies from our laboratory also indicated that carfilzomib retained appreciable activity against BTZ-resistant leukemia
[41, 52] and solid tumor cell lines
[23, 35, 49]; similar findings were observed with ONX 0912
, an orally- bioavailable analog of carfilzomib which provokes irreversible binding via the epoxyketone group to the active site Thr1 residue in the β5 subunit. This unique property, rather than reversible binding by boronate-based BTZ, may still facilitate inhibition of the catalytic activity in mutated β5, thereby resulting in retention of drug sensitivity. Since levels of immunoproteasome subunits were largely unaltered and no mutations were observed in the β5i subunit in JY/BTZ cells, it may also explain the marginal levels of cross-resistance that were observed for the immunoproteasome-specific inhibitor ONX 0914. In this context, it is worthwhile mentioning that recent crystal structure studies identified Met45 in β5i as a major determinant in the efficient docking of ONX 0914 to the catalytic binding site
. The Met45Ile mutation in the constitutive β5 subunit has apparently lesser consequences for ONX 0914 activity
. Together, constitutive and immunoproteasome targeted epoxyketone-based PIs like carfilzomib, currently evaluated in phase I-III clinical trials
, as well as ONX 0914 may readily overcome BTZ-resistance.
Besides antiproliferative effects, BTZ can also elicit potential anti-inflammatory effects by suppression of the release of pro-inflammatory cytokines through inhibition of NF-κB activation
[18–20, 25, 26]. In this respect, TNF-α has received the most attention as one of the critical cytokines in the pathophysiology of arthritis, and is thus a major druggable target for biological agents and small molecules
[20–22, 48]. Consistent with data from the Komano group
, we noted that JY/WT cells could produce appreciable levels of TNF-α, which could be partially inhibited by BTZ. Of additional interest was the notion that alongside these short term effects, acquisition of BTZ resistance in JY cells provoked a dramatic reduction in the basal levels of TNF-α production, being most pronounced in JY/100 cells. It remains to be established whether or not this effect is due to diminished response to activation stimuli, or aberrations in TNF-α secretion per se. In either case, prolonged BTZ exposure may elicit potential anti-inflammatory effects even when anti-proliferative effects are compromised.
Acquisition of BTZ resistance in JY cells was associated with opposite impacts on cell surface expression of two common B-cell surface markers: down regulation of CD19 and upregulation of CD20. CD19 is a transmembrane protein and co-receptor molecule that modulates signals through the B cell antigen receptor (BCR) to control B cell-differentiation from pro/pre-B cells to resting pre-B cells in bone marrow. Consistently, CD19−/− mice have exhibited defects at stages of early B cell development and reduced levels of circulating B-cells, as reviewed in Del Nagro et al.
. It is yet unclear whether reduced CD19 levels in JY/BTZ cells would result in a B cell differentiation arrest; this should be subject for further studies using panels of B cell differentiation-restricted cell surface markers.
CD20 serves as a Ca2+-permeable cation channel protein expressed on B cells from immature to mature stages of differentiation, as recently reviewed by Thaunat et al.
. BTZ has been reported to induce disregulation of intracellular Ca2+, triggering caspase activation and initiating apoptosis
. It is unknown whether this off-target effect of BTZ alters CD20 function and expression during resistance development. Rather, recent studies have pointed to the role of the ubiquitin proteasome system in the regulation of CD20 expression, as rituximab-resistant lymphoma cells have displayed upregulated expression of proteasome subunits
, and treating Raji human Burkitt lymphoma cells with BTZ for 24–48 hours has down-regulated CD20 expression
. In accordance with these studies, we now showed that impaired (poly) ubiquitination of proteins upon 24–48 hour BTZ treatment in resistant JY/BTZ cells attenuated degradation of CD20, resulting in a net 3-fold increase in CD20 expression in JY/100 cells and markedly enhanced rituximab-mediated CDC in JY/100 cells as compared to JY/WT cells. Based on the differential impact on CD20 expression in BTZ-sensitive and BTZ-resistant cells, one should take caution when considering combining rituximab and BTZ for B cell targeting; efficacy may only be realized in BTZ-resistant cells. Moreover, the current findings for JY lymphoblastic cells should also await further corroboration for B-cell neoplasms (e.g. mantle cell lymphoma) for which BTZ is a therapeutic option. Finally, it would be of additional future interest to explore whether aberrations in protein ubiquitination provoked by prolonged exposure to either BTZ or other types of PIs, may be instrumental to upregulated expression of other B cell surface markers that could elicit antibody-mediated CDC.