Enzymatic Activities Of Circulating Plasma Proteasomes (cProt) In Newly Diagnosed Multiple Myeloma (NDMM) Patients Undergoing Treatment With Carfilzomib, Lenalidomide and Dexamethasone (CRd)
Authors: Manasanch Elisabet E. , de Larrea Carlos Fernandez , Zingone Adriana , Korde Neha , Tageja Nishant , Bhutani Manisha , Kazandjian Dickran , Wu Peter , Costello Rene , Burton Debra , Carter George , Zuchlinski Diamond , Mulquin Marcia , Maric Irina , Calvo Katherine R , Yuan Constance , Stetler-Stevenson Maryalice , Roschewski Mark , Landgren Ola 
Published: October 2019

Abstract

Background
The proteasome inhibitor carfilzomib is highly effective in the treatment of multiple myeloma (MM). It selectively and irreversibly binds the chymotrypsin-like (CHYM) active site in the β5 subunit of the proteasome. One prior retrospective study based on 50 NDMM patients found maintained elevated total cProt levels post therapy to be associated with poorer response to therapy and shorter overall survival (PMID: 17095627). We conducted a study based on NDMM patients treated with carfilzomib, lenalidomide and dexamethasone (CRd). The aim of this investigation was to assess the role of specific enzymatic activities of plasma cProt in relation to treatment responses.

Methods
We assessed 37 newly diagnosed MM patients from our ongoing clinical trial using CRd therapy (NCT01402284). Baseline demographic, clinical and laboratory parameters were collected. Plasma samples were collected at time intervals as specified in the protocol [C1D1 (baseline), C1D2 (post single dose carfilzomib), C2D1, C4D1 and C8D1] and frozen to -80ºC. Patient responses were assessed per current IMWG guidelines. CHYM, caspase-like (CASP), and trypsin-like (TRYP) activities from cProt were assayed by continuously monitoring the production of 7-amino-4-methylcoumarin (AMC) from fluorogenic peptides in plasma. Briefly, plasma samples were activated with SDS (for chymotrypsin-like and caspase-like) or 10% Tween-20 (for trypsin-like). The reaction wells contained 30 μL assay buffer (25 mmol/L HEPES), 10 μL activated sample, and 10 μL of the prospective fluorogenic peptide-AMC substrate. To measure the fluorescence release of free AMC with time, the SpectraMax M5 (Molecular Devices) instrument was used with a read interval of 1 min during 30 min at 37ºC. Samples were analyzed per triplicate. Enzymatic activities were quantified (pmol AMC/s/mL plasma) by generating a standard curve of AMC. A two tailed Wilcoxon signed rank test was used for comparisons across matched cProt values. The Mann Whitney Test was used to compare differences between two independent groups of cProt values. Spearman’s rank correlation was used for correlation between cProt levels and clinical parameters. All p-values are two-tailed.

Results
Patients had a median age of 61 years (range 40-86; 57% males). Median M-spike was 2.7 (0.8-7.1) g/dL, isotypes included IgG (n=26), IgA (n=6), kappa (n=4) and lambda (n=1); International Staging System (ISS) Stage I (n=19) and II (n=18). Median C1D1 levels of chymotrypsin-like, caspase-like and trypsin-like activity for all patients were 0.84 (0.12-3.89), 0.85 (0.31-2.28) and 2.95 (1.4-7.39) pmol AMC/s/ml plasma, respectively. CHYM and CASP C1D1 levels correlated with the levels of baseline IgG (r=0.42, CHYM; r=0.38 CASP) and the levels of baseline β2-microglobulin (r=0.37, CHYM; r=0.45, CASP). Additionally, CHYM correlated with levels of free kappa (r=0.33) and levels of serum M-protein (r=0.34); and CASP correlated with the percentage of plasma cell infiltration in core bone marrow biopsy (r=0.36). Baseline TRYP levels did not correlate with clinical parameters. Median CHYM (1.3 versus 0.7 pmol AMC/s/ml plasma) and CASP (1.0 versus 0.7 pmol AMC/s/ml plasma) levels, but not TRYP levels, were were non-significantly elevated in Stage II ISS compared to Stage I patients. CHYM levels after an initial single dose of carfilzomib were reduced by a median of 68.2% (p<0.05), whereas CASP and TRYP levels were increased by 5.3% (p>0.05) and 0.4% (p>0.05), respectively. CASP and TRYP levels measured at C2D1, C4D1 and C8D1 remained stable throughout treatment cycles. When compared to baseline, median CHYM levels decreased significantly at C1D2 (after one dose of carfilzomib), then increased at C2D1 and steadily decreased again throughout treatment at the C4D1 and C8D1 time intervals.

Conclusions
In newly diagnosed MM patients, pre-treatment CHYM and CASP plasma activity levels were non-significantly higher among patients with higher ISS stage. Peripheral blood collected 24 hours after a single dose carfilzomib showed reduced CHYM levels by >60% in most patients. Fluctuations in CHYM, CASP and TRYP levels were noted throughout CRd treatment; no consistent patterns were observed in relation to response to therapy.

Disclosures:
No relevant conflicts of interest to declare.