PSMB5 mutations described for the first time in multiple myeloma patients

Proteasome inhibitors (PIs), such as bortezomib (BOR), carfilzomib (CARF) and ixazomib (IXA), are effective treatments for multiple myeloma (MM) patients (pts). PIs target the proteasome, rendering it dysfunctional. This results in accumulation and subsequent enhanced cell death of myeloma cells. Each PI targets different subunits of the proteasome. Similar to the resistance MM cells acquire to other treatments, they will eventually stop responding to PI treatment. Identifying specific mutations in MM cells that may confer resistance to PI treatments can help understand the biological mechanism of this resistance and find effective ways to overcome it.

In this study, Santiago Barrios and Thorsten Stühmer from Martin Kortüm’s group at the Department of Heamatology-Oncology, University Hospital Würzburg, Würzburg, Germany,

and collaborators discovered four single point mutations in the PSMB5 (proteasome subunit beta 5) gene identified in an MM pt refractory to bortezomib (BOR) treatment. Although mutations in the PSMB5 gene are known to confer resistance to long-term BOR treatment in MM cell lines, this is the first time these mutations are described in an MM pt.

Key Data:

• Data obtained from different Multiple Myeloma Mutation panel (M³P) cohorts and other published and unpublished datasets
• Total number of cases analyzed = 1596 with different treatment status (newly diagnosed, first relapse, refractory)
• Mutational analysis of proteasome subunit genes included = PSMA1, PSMB5, PSMB8, PSMD1, PSMG2
• Mutation frequency of proteasome genes increased with drug treatment: only one mutation in PSMB5 found among 1241 samples of newly diagnosed MM pts (0.08%); two PSMB5 mutations detected among 194 first relapse MM samples (1.0%); two additional PSMB5 mutations detected among 161 multi-refractory MM pts (1.2%)
• Four PSMB5 mutations detected at third relapse in a male Caucasian patient with λ light chain MM, heavily pre-treated with BOR
• Study Time Points (TP): Time point (TP) 1 = at diagnosis; TP2 = at first relapse after VTD-PACE [BOR, thalidomide, dexamethasone (DEX) – cisplatin, doxorubicin (A), cyclophosphamide and etoposide] treatment and first ASCT (autologous stem cell transplant); TP3 = at third relapse after VCD and second ASCT (second relapse) followed by four cycles of PAD (BOR, A and Dex) in combination with lenalidomide (LEN); TP4 = disease progression after three cycles with PAD-Pomalidomide (POM); TP5 = disease progression after two additional cycles of POM-Elotuzumab-DEX; TP6 = patient treated and responsive to KRD (CARF, LEN, and DEX)
• Four PSMB5 mutations were detected after initiation of PAD-POM treatment at TP3
• PSMB5 mutations are as follows: c.235G>A (p.A20T), c256G>C (p.A27P), c.312G>C (p.M45I), and c.365G>A (p.C63Y) (protein positions refer to the cleaved mature protein); variant read (VR) = 1.9-5.3%; all located in a highly conserved region in exon 2
• Four months later (TP4): M45I and A20T, detectable; A27P and C63Y, undetectable in the targeted M³P sequencing
• Three months later (TP5): A27P and C63Y remained undetectable
• Nine months later (TP6): none of the four mutations were detectable
• Retrospective analysis at TP1 (diagnosis) and TP2 (first relapse) showed that c.235G>A and c.365G>A were already present
• The PSMB5 mutations were present in independent subclones
• VR frequencies of single mutations: A20T = 3.2%; A27P = 2.3%; M45I = 5.3%; C63Y = 1.9%
• VR sum of all subclones with a PSMB5 mutation: 25% of MM cells sampling at TP3
• Structural location of mutations: A20T, A27P, and M45I are located within the S1 pocket, near/at the binding site for BOR; C63Y is located in proximity to the substrate-binding channel
• Functional analysis of the PSMB5 mutations were performed by transfecting the AM0-1 and L363 MM cell lines with wild-type or mutant PSMB5 plasmids
• After PSMB5 wild-type and mutant overexpression, AMO-1 and L363 cell survival was examined in cells treated with different doses of BOR, IXA, and CARF
• Overexpression of mutant PSMB5 resulted in pronounced resistance against BOR; similar resistance to IXA as with BOR, although at higher effective concentrations; resistance to CARF for the A20T and M45I mutations but a  or absent effect in the C63Y and A27P mutants; CARF resistance was easily overcome with increasing drug concentrations
• To evaluate the effect of PI treatment on the activity of proteasome subunits (β1, β1i, β2, β2i, and β5), a set of activity-based probes (ABP) was used to visualize proteasome activities
• BOR and IXA: showed similar effects on β5 and β1/β1i subunits in the AMO-1 cell line overexpressing wild-type PMSB5; lost inhibitory effect of β5 when PMSB5 mutations were present
• CARF: The most specific β5 subunit inhibitor in the ABP assay, with hardly any activity on the β1/β1i subunits

Conclusions

This is the first study detecting somatic mutations from MM pts in the PSMB5 gene. Furthermore, the functionality of the mutations and resistance to different PIs was validated. The increased incidence of mutations in proteasome subunit genes observed alludes to a possible mechanism conferring resistance to PIs in MM pts.