Amin Sobh

Glucocorticoids (GCs) coordinate immunity, inflammation, and metabolism through allosteric regulation of the glucocorticoid receptor (GR) transcription factor. GCs are indispensable anti-inflammatory drugs yet linking specific ligand–receptor structural states to specific biological outcomes has remained a major barrier to designing safer, more selective therapies. Using structure-based design, we developed selective glucocorticoid receptor modulators (SGRMs) of immune function by extending a steroidal scaffold from the ligand-binding pocket into an adjacent solvent channel. These SGRMs suppressed T cell pro-inflammatory cytokines and promoted differentiation of memory precursor T cells while showing minimal induction of M2 macrophage polarization or T cell checkpoint proteins PD-1 and CTLA-4, all key targets of immunotherapy. Molecular dynamics simulations revealed that solvent-channel substituents function as a lever arm to drive dynamic oscillations in the steroid core, thereby allosterically tuning GR activity states. Systematic perturbation of immune cells with a graded series of ligands enabled a l igand p erturbation with m achine l earning (LPML) framework to map coregulated responses across cell types and identified effector T cell gene networks tightly coupled with immune checkpoint induction. This approach outlines a general strategy for decoding the logic of allosteric drug action, enabling the rational design of SGRMs with tailored immunomodulatory profiles.

Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2). A genome-wide CRISPR screen in MM cells identified adenylate kinase 2 (AK2), an enzyme critical for high-energy phosphate transfer from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased nicotinamide adenine dinucleotide phosphate (NADP[H]) critical for conversion of ribonucleotides to deoxyribonucleosides, leading to replication stress, DNA damage, and apoptosis. Driving a large genome-wide increase in chromatin methylation, NSD2 overexpression depletes S-adenosylmethionine, compromising the synthesis of creatine from its precursor, guanidinoacetate. Creatine supplementation restored NADP(H) levels, reduced DNA damage, and rescued AK2-deficient t(4;14) MM cells. As the creatine phosphate shuttle constitutes an alternative means for mitochondrial high-energy phosphate transport, these results indicate that NSD2-driven creatine depletion underlies the hypersensitivity of t(4;14) MM cells to AK2 loss. Furthermore, AK2 depletion in t(4;14) cells impaired protein folding in the endoplasmic reticulum, consistent with impaired use of mitochondrial adenosine triphosphate (ATP). Accordingly, AK2 suppression increased the sensitivity of MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.

Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.

Control cells, treated only with biotin

Supplementary Methods, figures and tables

Cells pre-treated for 3h with 10 µM brefeldin A

Cells pre-treated for 3h with 20 µM nelfinavir.

All identified binding partners of nelfinavir across multiple cell lines

Cells pre-treated for 3h with 10 µM brefeldin A

All identified binding partners of nelfinavir across multiple cell lines