Pharmacological Targeting of IRF4 As a Therapeutic Strategy for Multiple Myeloma
Authors: Agius Michael P. , Hevenor Laura , Payne N Connor , Liu Qi , Seo Hyuk-Soo , Sklavenitis-Pistofidis Romanos , Heilpern-Mallory Daniel , Bashyal Puspalata , Aranha Michelle P. , Lightbody Elizabeth D. , Mazitschek Ralph , Dhe-Paganon Sirano , Qi Jun , Ghobrial Irene 
Published: December 2024

Introduction
Interferon regulatory factor 4 (IRF4) is an oncogenic transcription factor (TF) in several hematological malignancies. In multiple myeloma (MM), IRF4 serves as an MM-specific regulator of cell survival, making it an ideal target for therapy development. However, to date, no pharmacological agents have been successfully developed to directly target IRF4 due to the challenging nature of directly targeting TFs. Moreover, no probes or screening-based assays have been developed for IRF4 or any member of the IRF family. IRF4 is a top-ranked dependency of MM, activated B-cell-like lymphoma, and leukemia. Moreover, IRF4 is a validated clinical target given the success of IMiD-based regimens. Thus, orally available small molecules that can directly target IRF4 would significantly benefit patients with hematological malignancies. To this end, we selected the IRF4 interferon accessory domain (IAD), and specifically the SPI1 (or PU.1) binding pocket, to design and develop the first small molecule degrader against IRF4.
Method and Results
To identify chemical ligands, we developed a series of biochemical and cellular assays for high-throughput screening. We validated the most potent hit, compound H1, as a binder of both recombinant IAD domain and cellular IRF4 with biophysical and in-cell target engagement assays. Additionally, we developed an in-cell BRET-based (bioluminescence resonance energy transfer) assay to evaluate the engagement of our compound series against IRF4 in HEK293T cells. One of H1's enantiomers, (S)-H1,had an IC50 of 0.3 µM, and its counter partner (R)-H1 shows no binding (&gt;100 µM) by TR-FRET. Next, we successfully solved the first X-ray structure of the human IRF4 IAD domain at 2.5 Å to assist our structure-based drug design efforts of more potent binders. To reproduce the strong anti-myeloma effects of IRF4 knockdown or knockout, we turned our novel binder into a proteolysis targeting chimera (PROTAC) of IRF4 that can selectively remove IRF4 in MM cell lines. We linked (S)-H1 to E3 ligase ligands of cereblon to create the very first IRF4 degrader, dIRF4, which can induce proteasomal degradation of IRF4 selectively and has strong cytotoxic effects in all myeloma lines evaluated in vitro. We synthesized inactive controls derived from dIRF4, using a (R)-H1 or an inactive IMiD ligand, and showed the proteasomal and cereblon requirement needed for IRF4 depletion. Using RNA sequencing and proteomic workflows, we showed that dIRF4 has on-target effects of IRF4 depletion and significantly down-regulates IRF4-controlled pathways in MM, including PIM2, at the transcript and protein level. Finally, early in vivo pharmacokinetic experiments show favorable Cmax (4500ng/mL) and toxicity profiles after intraperitoneal administration, warranting dIRF4 use in in vivo studies.
Conclusions
Our results have led to the development of dIRF4, the first pharmacological agent directly targeting an oncogenic transcription factor in myeloma or any IRF family transcription factors. Our results support the clinical development of an IRF4 degrader in myeloma. Furthermore, we demonstrate a feasible developmental strategy to target the IRF family by leveraging their IAD domains, including IRF8, which can be chemically targeted by this approach and widen the therapeutic landscape in hematological cancers.