(PA-292) Development of Novel CREB1-PROTACs for Enhanced Targeting of CREB1 in Multiple Myeloma

Multiple myeloma (MM) cells originate from antibody-producing plasma cells and endure chronic oxidative and proteotoxic stress due to the excessive immunoglobulin and free light chain production. We previously demonstrated that CD56 (neuronal cell adhesion molecule 1) promotes CREB1 (cAMP responsive element binding) activation in MM cells to support survival, though its precise mechanism of action remains unclear (Cottini et al, 2022).

Methods:

A panel of MM cell lines were treated with DMSO, CREB1 inhibitor (666-15), CREB1-PROTACs (Proteolysis-targeting chimeras), and proteasome inhibitors at variable concentrations. CREB1 loss-of- and gain-of-function models were also established.

Results:

Patients with high CREB1 expression had increased activation of gene sets associated with immune escape, leading to elevated expression of HLA-E (Ismael et al, 2024), as well as gene sets linked to oxidative stress endurance and modulation of the unfolded protein response (UPR). Silencing CREB1 with shRNAs in OPM-2 and H929 cells increased total and mitochondrial reactive oxygen species levels. CHIP-sequencing analysis revealed that CREB1 directly binds to the promoters of NFE2L2 (NRF2) and EIF2AK3 (PERK). Overexpression of CREB1 in U266 cells upregulated these factors, inducing the expression of genes involved in oxidative and protein stress adaptation. In contrast, CREB1 silencing in OPM-2 and H929 cells led to the downregulation of PERK, MCL1, BCL2, and TXNIP, while increasing apoptotic markers such as DDIT3 (CHOP), and PPP1R15A (GADD34). We then confirmed that the CREB1 inhibitor 666-15 downregulated CREB1 targets, including PERK, BCL2, MCL1, and TXNIP.

Building on 666-15 backbone, four distinct CREB1-PROTACs were synthetized by the Medicinal Chemistry Core at the Ohio State University. PROTACs are a novel class of drugs which directly eliminate target proteins via the ubiquitin-proteasome system. Unlike conventional inhibitors that bind to an active site, PROTACs require only an anchoring point on the protein, enabling the targeting of previously “undruggable” proteins.

We tested these four compounds for their ability to degrade CREB1 and induce cell death. All compounds successfully induce CREB1 degradation at various time points and concentrations, with compound #28 proving the most potent. Treatment of MM cell lines with 50–500 nM of compound #28 reduced viability and induced apoptosis while decreasing CREB1 downstream targets, including TXNIP, HLA-E, MCL1, and BCL2.

Since PROTACs require active proteasomes, they cannot be combined with bortezomib or carfilzomib. Instead, we explored combination strategies with immunomodulatory drugs, observing synergy between compound #28 and lenalidomide, pomalidomide, and iberdomide. Further mechanistic studies, along with drug metabolism and pharmacokinetic analyses, are ongoing.

Conclusions:

In conclusion, CREB1-PROTACs represent a promising new class of compounds for the treatment of MM, warranting further development and investigation.