Faculty Bibliography

Plasma cells producing high levels of paraprotein are dependent on the unfolded protein response (UPR) and chaperone proteins to ensure correct protein folding and cell survival. We hypothesized that disrupting client-chaperone interactions using heat shock protein 90 (Hsp90) inhibitors would result in an inability to handle immunoglobulin production with the induction of the UPR and myeloma cell death. To study this, myeloma cells were treated with Hsp90 inhibitors as well as known endoplasmic reticulum stress inducers and proteasome inhibitors. Treatment with thapsigargin and tunicamycin led to the activation of all 3 branches of the UPR, with early splicing of XBP1 indicative of IRE1 activation, upregulation of CHOP consistent with ER resident kinase (PERK) activation, and activating transcription factor 6 (ATF6) splicing. 17-AAG and radicicol also induced splicing of XBP1, with the induction of CHOP and activation of ATF6, whereas bortezomib resulted in the induction of CHOP and activation of ATF6 with minimal effects on XBP1. After treatment with all drugs, expression levels of the molecular chaperones BiP and GRP94 were increased. All drugs inhibited proliferation and induced cell death with activation of JNK and caspase cleavage. In conclusion, Hsp90 inhibitors induce myeloma cell death at least in part via endoplasmic reticulum stress and the UPR death pathway.

Recent evidence suggests a role for natural killer (NK) cells in the control of multiple myeloma. We show that expression of the NK cell receptor DNAM-1 (CD226) is reduced on CD56(dim) NK cells from myeloma patients with active disease compared with patients in remission and healthy controls. This suggested that this receptor might play a role in NK-myeloma interactions. The DNAM-1 ligands Nectin-2 (CD112) and the poliovirus receptor (PVR; CD155) were expressed by most patient myeloma samples analyzed. NK killing of patient-derived myelomas expressing PVR and/or Nectin-2 was DNAM-1 dependent, revealing a functional role for DNAM-1 in myeloma cell killing. In myeloma cell lines, cell surface expression of PVR was associated with low levels of NKG2D ligands, whereas cells expressing high levels of NKG2D ligands did not express PVR protein or mRNA. Furthermore, NK cell-mediated killing of myeloma cell lines was dependent on either DNAM-1 or NKG2D but not both molecules. In contrast, the natural cytotoxicity receptor NKp46 was required for the killing of all myeloma cell lines analyzed. Thus, DNAM-1 is important in the NK cell-mediated killing of myeloma cells expressing the cognate ligands. The importance of NKp46, NKG2D, and DNAM-1 in myeloma killing mirrors the differential expression of NK cell ligands by myeloma cells, reflecting immune selection during myeloma disease progression.

Increases in free light chain (FLC) production are associated with disease progression in multiple myeloma (MM). Using a double immunofluorescence staining method to produce a differential count of plasma cells in bone marrow, single populations were demonstrated, containing intact monoclonal immunoglobulins (M-Igs) in 74% and FLCs only in 8% of cases. However, 18% contained a mixture of both cell populations. Progression from cells making intact M-Ig to cells restricted to FLC only production occurred in individual cases during the course of their disease. The presence of FLC only cells was associated with shortened survival.

The combination of bortezomib (velcade), pulsed dexamethasone and weekly cyclophosphamide (CVD) in relapsed/refractory myeloma patients induces high overall (75%) and complete (31%) response rates compared to velcade/dexamethasone (overall 47%, CR 5%) and velcade alone (overall 27%, CR 0%). The toxicity profiles including thrombocytopenia, neutropenia, and neuropathy were comparable between the groups.

A greater understanding of the biology of myeloma has focused research on the identification of novel target-based treatment strategies. Proteasome inhibition represents one such approach and the introduction of bortezomib, the first-in-class proteasome inhibitor, has been a major breakthrough in the treatment of multiple myeloma. As a result of its novel mechanism of action, bortezomib has been shown to induce responses in patients previously refractory to treatment, and results in increased progression-free and overall survival rates. The current understanding of the biology of the proteasome and the mechanism by which proteasome inhibition leads to myeloma cell death is described in this review. The role of proteasome inhibitors in the management of myeloma is also discussed.

Polymorphisms in NFKBIA may be important in pre-disposition to and outcome after treatment, of multiple myeloma (MM). The NFKBIA gene product, IkappaBalpha, binds to NF-kappaB preventing its activation and is important in mediating resistance to apoptosis in B-cell lymphoproliferative diseases. This study investigates eight polymorphisms across the NFKBIA gene in large patient and control populations. Significant differences in the frequency of particular polymorphisms were noted between patients and controls. A risk haplotype [GCCTATCA] for MM was also identified (P=0.006). Analysis of the genetics of NFKBIA may lead to associations with disease progression and survival and thus more personalized therapy.

A retrospective case-matched study was conducted to compare the oral regimen CTD (cyclophosphamide - thalidomide - dexamethasone) and infusional CVAMP (cyclophosphamide - vincristine - doxorubicin - methylprednisolone) as induction therapy followed by autologous peripheral blood stem-cell transplantation (PBSCT) for newly diagnosed multiple myeloma patients. The response rate after three cycles of treatment was statistically higher with CTD (n = 27) compared to CVAMP (n = 27) (89% vs. 56%, P = 0.016). Toxicity studies showed more neutropenia (grade 3/4) (4% vs. 60%, P = 0.0002) with CVAMP and more thrombotic episodes with CTD (11% vs. 4%). CTD may emerge as the superior induction regimen prior to PBSCT, in terms of high efficacy and better tolerability.

Alemtuzumab has been proposed as a therapeutic agent in myeloma. CD52 was detected on plasma cells in 46/106 patients but levels were 30-fold lower than on alemtuzumab-responsive cells (n=138) and 8-fold lower than on alemtuzumab-resistant cells (n=57). The data suggest that myeloma plasma cells are unlikely to be depleted by alemtuzumab in most patients.