Unraveling Proteasome Inhibitor-Induced Cardiovascular Toxicity through Integrative Multiomics

Stanford University, Stanford CA

Investigators

Abstract

PROJECT SUMMARY Proteasome inhibitors (PIs) represent novel cancer therapeutics highly effective in treating hematologic malignancies and have remarkably improved cancer survival rates. However, the utilization of PIs, particularly carfilzomib (CFZ), is largely hampered by substantial cardiovascular toxicity, which occurs through poorly characterized mechanisms. In this proposal, we aim to leverage a humanized multiple myeloma (MM) mouse model, human induced pluripotent stem cells (iPSCs), CRISPR interference and activation (CRISPRi/a) screens, single-cell RNA sequencing (scRNA-seq), deep learning, and RNA splicing biology to elucidate the molecular signatures underlying CFZ-induced cardiovascular dysfunction. We will focus on investigating the role of pathogenic alternative splicing events (ASEs) in smooth muscle cells (SMCs) and endothelial cells (ECs) in both mice and iPSCs. Finally, we will employ antisense oligonucleotides (ASOs) targeting candidate pathogenic ASEs to evaluate their effectiveness in preventing or mitigating CFZ-induced cardiovascular toxicity. In Aim 1, we will confirm CFZ-induced cardiovascular dysfunction phenotypes (e.g., hypertension, microcirculation dysfunction, and cardiac hypertrophy) in an MM mouse model and collect vehicle- and CFZ-treated mouse aortas for scRNA- seq. We will then perform extensive alternative splicing analysis using two novel algorithms, SpliZ and SpliceAI, developed by our team. In Aim 2, we will differentiate pooled iPSC lines derived from healthy donors and CFZ- treated cancer patients with or without cardiovascular phenotypes (5M+5F/group) into iPSC-SMCs and iPSC- ECs and construct 3D-engineered vascular tissues (iPSC-EVTs). We will select species-conserved, CFZ- induced differentially expressed splicing factors predicted to be pathogenic to conduct CRISPRi/a screens in pooled iPSC-EVTs and identify causal genes downstream of candidate splicing factors. In Aim 3, we will employ minigene splicing assays to confirm putative pathogenic ASEs in identified causal genes and design specific ASOs to block their activity. Therapeutic efficacy of ASOs will be validated first in primary and iPSC-derived EVTs. Promising candidate ASOs will undergo further validation in MM mice to evaluate their rescue effects on CFZ-induced hypertension, thrombosis, arrhythmia, and cardiac dysfunction. We anticipate that the successful completion of these studies will provide new mechanistic insights into CFZ-induced cardiovascular dysfunction and facilitate the development of novel therapeutics, enabling cancer patients to safely receive life-saving PI- based therapies. Moreover, this proposal will contribute novel insights into the role of aberrant alternative splicing in vascular pathology beyond the scope of cardio-oncology.