The Deubiquitinating Enzymes Of The 19S Proteasome Offer Novel Therapeutic Opportunity In Bortezomib Resistant Waldenströms Macroglobulinemia

The ubiquitin proteasome system (UPS) maintains homeostasis in eukaryotic cells by degrading and recycling proteins associated with DNA transcription and cell cycle regulation. In tumor cells with a high protein turnover rate, particularly in immunoglobulin secreting cancers such as Waldenströms Macroglobulinemia (WM), proteasomes are critical for sustained cell proliferation. Proteasome inhibitors (PI) such as bortezomib and carfilzomib target the B5 subunit of the proteasome catalytic core and disrupt its activity leading to accumulation of polyubiquitinated proteins. Although highly effective in WM, persistent use of bortezomib eventually leads to emergence of a bortezomib resistant (BR) subclone. Point mutation in the PSMB5 gene (encoding the B5 subunit) leading to altered binding kinetics is identified as a potential underlying mechanism of PI resistance. Despite these adaptive modifications that support resistance to PI resistance, the UPS as a whole remains a relevant target with vulnerable sites that can be therapeutically exploited. The 19S cap of the proteasome contains deubiquitinating enzymes (DUBS) that function to deubiquitinate proteins prior to their entry into the proteasomal core. Using a novel DUB-inhibitor b-AP15, which blocks the UCHL5 and USP14 DUBS, D’Arcy et al demonstrated that disruption of these DUBS does not alter proteasomal activity yet induces robust tumor cell death in various cancers.

Investigation of b-AP15 in preclinical models of BR WM and characterization of associated downstream signaling interactions.

For these studies, we developed and used BR variants of the most commonly used WM cell lines (variants termed BCWM.1/BR, RPCI-WM1/BR and MWCL-1/BR). Proteasomal activity was measured in these cells using synthetic fluorogenic peptide substrates and apoptosis was measured by annexin-v/PI staining. For gene expression profiling (GEP), the Nanostring nCounter mRNA quantification assay was utilized.

We treated all BR WM clones with either single agent bortezomib (10nM), carfilzomib (10nM), b-AP15 (100nM) or all three agents in combination and assessed their effect on chymotrypsin-like, caspase-like and trypsin-like proteasomal activity. b-AP15, either alone or in combination with the other PI, did not alter proteasomal activity, nor did it abrogate the inhibition of chymotrypsin-like activity of other PI. Although proteasomal activity was not disrupted, we observed an increase in high molecular weight ubiquitinated proteins on immunoblot analysis. We hypothesized that despite the BR cells resistance to B5-targeting PI, they would remain sensitive to UPS inhibition if targeted through DUB inhibition. We treated BR cells with b-AP15 at concentrations ranging from 50nM - 1.25uM and observed that b-AP15 induced robust WM cell death in a dose and time dependent manner. Apoptosis was more pronounced in MWCL-1/BR cells and optimal cell death (55%) was observed at 500nM; however a commitment towards cellular demise was evident by PARP-1 and caspase cleavage as early as 3 hrs. Use of a pan-caspase inhibitor resulted in rescuing of the BR WM cells from b-AP15 mediated death and demonstrated a caspase-dependent death process. In order to gain further mechanistic insight into the processes that regulates response to b-AP15, we performed GEP and immunoblot of BR WM cells treated with 50nM of b-AP15. We observed an increase in phospho-p38 protein, which belongs to the MAPK family and regulates IL8 secretion. When BR WM cells were treated with a p38 inhibitor in combination with b-AP15, a significant decrease in cell viability was noted relative to b-AP15 (100nM) alone. Furthermore, GEP and network analysis identified key regulator genes associated with p53, cell cycle and MAPK signaling.

We provide first evidence that inhibition of the DUBS, UCHL5 and USP14 with b-AP15 results in caspase-dependent apoptosis of BR WM cells. Our investigation uncovers important molecular interactions that may dictate the BR cells responsiveness to DUB-inhibition by b-AP15. These observations validate the pervasive role of the UPS and confirm alternate therapeutics targets using our unique BR WM models.

We are grateful to the Waterfall Waldenström Macroglobulinemia Research Fund, the Leukemia and Lymphoma Society and the International Waldenström Macroglobulinemia Foundation for their continued support.

Martin:Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau.