Abstract
Introduction: Despite the promise of B-cell receptor-associated kinase inhibitors (BCRi) in CLL, resistance to these agents is inevitable. Ubiquitin-proteasome systems are altered in cancer, leading to destabilization of tumor suppressors, overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. Neoplastic B cells exhibit a state of heightened cellular stress and are thereby susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The UPR is activated in CLL cells upon sIgM signaling and inhibited by ibrutinib. Proteasome inhibitors demonstrate clinical activity in certain types of B-cell neoplasia but are inactive in CLL. Here, we investigated an alternative approach to harness the pro-apoptotic UPR in CLL by using TAK-243, a first-in-class small molecule UAE inhibitor.
Methods: Peripheral blood cells were obtained from patients with CLL (N=20) and isolated using Ficoll-Hypaque techniques. TAK-243 was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). TAK-243 efficacy was assessed in CLL cells and 10 DLBCL (diffuse large B-cell lymphoma) cell lines.
Results: TAK-243 induced ER stress and the UPR in CLL cells, followed by rapid apoptosis within 2 hours. Following 24-hour incubation, we established an IC50 of ~50 nM (Annexin V+ cells) in CLL cells. By contrast, primary B cells and T cells were less sensitive to TAK-243. Given the importance of tumor microenvironment in CLL cell survival, we evaluated the effect of TAK-243 in a CD40L-expressing stromal co-culture model. Whereas CD40L-stimulated CLL cells were resistant to BCRi, they were fully sensitive to UAE inhibition. TAK-243 had a similar IC50 (~50 nM) across DLBCL cell lines, independent of cell of origin.
Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and degradation of proteins controlled by the UPS in CLL cells and DLBCL cell lines. UPR induction occurred within 2 hours, as shown by activation of eIF2α (in both CLL and DLBCL cells) and oligomerization and autophosphorylation of PERK (in DLBCL cells). After 4 hours, neoplastic B cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34, and NOXA. These events were accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of Mcl-1 and, ultimately, cleavage of caspase-3 and PARP. TAK-243 inhibited NFκB pathway, as shown by accumulation of IκBα, a negative pathway regulator. The extent of the UPR in CLL cells varies depending on the initiating signal. For example, B-cell receptor crosslinking induced expression of CHOP and GRP78 in CLL cells, but only weak activation of PERK and no IRE1-dependent processing of XBP1 (Krysov S, et al. Blood. 2014). Targeting UAE in CLL cells induced robust activation of eIF2α, upregulation of CHOP, GADD34 as well as NOXA mRNA, indicating high sensitivity to this pathway.
TAK-243 induced a more rapid UPR and exhibited lower IC50 compared with the proteasome inhibitor bortezomib in CLL and DLBCL cells. While both drugs induced autophagy as shown by LC3 processing, only bortezomib treatment led to p62 degradation, suggesting that autophagy was inefficient in response to TAK-243 due to lack of ubiquitin conjugation. Our findings were confirmed in a mouse lymphoma xenograft model. OCI-LY3 cells were inoculated subcutaneously in the right flank of NSG mice and treatment with TAK-243 (10 or 20 mg/kg IV twice weekly) or vehicle control began when tumors reached 10 mm in size. Treatment led to reduced tumor progression, induction of ER stress, and decreased cell proliferation and survival.
Conclusions: The UAE inhibitor TAK-243 induces ER stress and promotes apoptosis in CLL cells in vitro and restricts lymphoma growth in vivo. TAK-243 exhibited greater in-vitro cytotoxicity in lymphoma cells compared to bortezomib. Targeting UAE is a novel approach to disrupt the UPS which may hold promise in therapy of CLL and other B-cell malignancies.
Disclosures
Spurgeon: Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:TG Therapeutics: Consultancy; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Takeda Oncology: Research Funding.
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