Kurylo, K. | bioRxiv Science

bioRxiv2026

Discovery and characterization of small molecule inhibitors of CBL-B that act as intramolecular glue to enhance T-cell anti-tumor activity

CBL-B is a RING-type E3 ubiquitin ligase that acts as a critical negative regulator of T-cell activation. It promotes T-cell anergy and suppresses immune responses through ubiquitin-mediated control of signaling proteins at the immunological synapse. T cells deficient in CBL-B activity lose their dependence on CD28 co-stimulation, exhibit heightened activation and increased cytokine production, and fail to re-establish anergy. In addition, mice deficient in CBL-B activity reject tumors. Together, this cellular mechanism and in vivo phenotype suggest inhibition of CBL-B may be a viable immuno-oncology therapeutic strategy. Here, we report the rational design and execution of a high-throughput screen (HTS) to identify small molecule inhibitors of CBL-B. This campaign led to the discovery of a scaffold that inhibits CBL-B E3 ligase activity with micromolar potency. Structural characterization revealed an intramolecular glue mechanism, in which the compound stabilizes the closed state of CBL-B, preventing phosphorylation of a tyrosine residue that is critical for activation and E2 binding. Iterative structure-activity optimization yielded compounds with nanomolar activity that enhanced T-cell activation and cytokine secretion in primary human T cells and suppressed tumor growth in a syngeneic colorectal mouse model. Together, these studies validate the biological rationale for pharmacological CBL-B inhibition and enabled the de novo discovery of intramolecular CBL-B glue inhibitors. This work culminated in the identification of NX-1607, a first-in-class oral CBL-B inhibitor now in clinical development for cancer immunotherapy.

immunology↗

bioRxiv2021

TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

A hallmark pathological feature of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function.

neuroscience↗