Despite the promise of targeting B-cell receptor-associated signaling kinases in indolent lymphomas, new agents in this class show limited activity in DLBCL, which remains an unmet clinical need. Ubiquitin-proteasome systems (UPS) are altered in cancer, leading to destabilization of tumor suppressors (e.g., TP53), overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. We and others have shown that neoplastic B-cells exhibit a state of heightened cellular stress and thereby are susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Proteasome inhibitors (e.g., bortezomib) have demonstrated clinical activity in B-cell neoplasia. Our group has previously targeted proximal components of the UPS in B-cell neoplasia by disrupting the Nedd8 pathway with pevonedistat, which inhibits the function of a subset of E3 ubiquitin ligases known as cullin-ring ligases. Here we investigated an alternative approach to targeting the UPS in DLBCL using TAK-243, a first-in-class small molecule UAE inhibitor. By inhibiting UAE, TAK-243 blocks ubiquitin conjugation, disrupting monoubiquitin signaling as well as global protein ubiquitination. Here we demonstrate that UAE inhibition effectively kills DLBCL cells via induction of ER stress.
Experiments were conducted in activated B-cell (ABC)-type (OCI-LY3, U2932) and germinal center (GC)-type (OCI-LY19, SUDHL4/10/16, VAL) DLBCL cell lines, and primary neoplastic B-cells derived from peripheral blood and lymph nodes of patients with CLL and DLBCL, respectively. CD40L-expressing stromal cells were used to induce NFκB activation in primary cells. TAK-243 was obtained from Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA).
TAK-243 induced ER stress and the UPR , followed by rapid apoptosis, as evidenced by PARP cleavage within 2 h, in both DLBCL cell lines and primary neoplastic B-cells. 24 h drug treatment yielded an IC50 of ~50 nM, as estimated by Annexin V expression. TAK-243 induced apoptosis of DLBCL cell lines independent of cell-of-origin (ABC- vs. GC). By contrast, primary T cells were significantly less sensitive to TAK-243.
Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and subsequent degradation of proteins controlled by the UPS in neoplastic B-cells. TAK-243-induced UPR occurred within 2 h, as evidenced by oligomerization and autophosphorylation of PERK, induction of BiP/GRP78 and activation of eIF2α. After 4 h of treatment, cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34 (10-fold) and NOXA (4-fold), accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of MCL-1, activation of BAK, and ultimately cleavage of caspase-3 and PARP.
TAK-243 inhibited both canonical and non-canonical NFκB pathways in neoplastic B-cells, as evidenced by accumulation of IκBα, a negative pathway regulator, and diminished p100/p52 processing, respectively. We and others have shown that CD40L induces NFκB and promotes resistance to spontaneous and drug-induced apoptosis in primary neoplastic B-cells. By contrast, CD40L-stimulated cells were fully sensitive to TAK-243 (IC50=50 nM). Furthermore, TAK-243 showed lower IC50 compared to bortezomib in DLBCL cell lines and primary samples, and induced accelerated and more pronounced ER stress and NFκB inhibition.
We observed increased MYC mRNA transcript and protein expression in neoplastic B-cells following treatment with TAK-243. DLBCL cells expressing high baseline MYC levels (by protein expression) showed enhanced susceptibility to TAK-243-mediated apoptosis compared with MYC-low cells. Engineered expression of MYC sensitized OCI-LY3 cells to TAK-243, while siRNA-mediated downregulation of MYC resulted in decreased apoptosis. It has been suggested that MYC may sensitize cells to apoptosis through the extrinsic pathway. Indeed, we found that TRAIL ligand sensitized DLBCL cell lines to TAK-243.
Thus, the UAE inhibitor TAK-243 induces ER stress, abrogates NFκB pathway activation and promotes apoptosis in DLBCL cell lines and primary neoplastic B-cells. Its in vitro activity compares favorably with bortezomib. Targeting UAE is a novel approach to disrupt the ubiquitin-proteasome system which may hold promise in therapy of B-cell malignancies.
Berger: Takeda Pharmaceuticals International Co.: Employment.
Asterisk with author names denotes non-ASH members.