Biology subjects

Belizaire, R.

Publications and source records attributed to Belizaire, R..

5 recordsLinked to original sources

bioRxiv2026

Targeting CBL ubiquitin ligase activation to downregulate tyrosine kinase signalling

The CBL E3 ubiquitin ligase is a critical regulator of tyrosine kinase (TK) signalling. CBL activity is regulated by a feedback loop in which an active TK phosphorylates CBL, relieving its autoinhibited conformation, and allowing ubiquitination of substrates, including TKs, leading to their degradation. Binding of the Src Like Adapter Protein 2 (SLAP2) to CBL can also activate autoinhibited CBL and promote substrate ubiquitination. Using an engineered CBL mutant in the SLAP2 binding interface, termed RE CBL, that mimics activation by SLAP2 binding, we characterized the cellular functions of this CBL activation mechanism. Comparison of wildtype and RE CBL interactomes using MiniTurboID showed extensive and overlapping interaction networks, with a discrete subset of proteins, including the known substrate, epidermal growth factor receptor (EGFR), as well as endocytic factors such as EPS15, in higher abundance with RE CBL compared to wildtype. Consistent with these observations, RE CBL interacted more readily with EGFR, enhanced EGFR internalization, and attenuated downstream signalling compared to WT. In Cbl null hematopoietic cells, RE CBL expression reduced sensitivity to cytokines IL-3 and GM-CSF, and decreased activation of the Src-family kinase Lyn. Furthermore, we optimized and conducted a small molecule screen to identify a group of structurally related compounds that, like SLAP2 binding, promoted CBL activation in vitro. Together these findings provide proof of concept for targeting CBL activity to downregulate TK signalling.

biochemistry↗

bioRxiv2025

Discovery of A Small Molecule non-IMiD Degrader of ZBTB7A for the Treatment of β-hemoglobinopathies

Sickle cell disease and {beta}-thalassemia, two major {beta}-hemoglobinopathies, pose significant clinical challenges globally. Current treatments often face limitations in efficacy and tolerability. The transcription factor ZBTB7A has emerged as a promising therapeutic target for reactivating fetal hemoglobin expression. Here, we report the discovery and characterization of SH6, a small molecule non-IMiD degrader of ZBTB7A. SH6 induces fetal hemoglobin in erythroid cell lines in a CRBN and ZBTB7A-dependent manner, and it is capable of inducing fetal hemoglobin expression in healthy donor, SCD and {beta}-thalassemia patient CD34+ cell derived erythroid cells. The efficacy of SH6 is confirmed in a xenotransplantation humanized mouse model. SH6 outperforms currently available therapeutic agents in vitro, and shows synergy with hypomethylating agents. SH6 exhibits a favorable in vivo toxicity profile. Our findings establish SH6 as a promising therapeutic lead candidate for further optimization towards clinical development for treatment of sickle cell disease and {beta}-thalassemia.

cell biology↗

bioRxiv2025

TTLL4 glutamyltransferase is a therapeutic target for NPM1-mutated acute myeloid leukemia

NPM1-mutated acute myeloid leukemia (AML) is defined by aberrant cytoplasmic localization of the mutant NPM1c protein, and therapeutic strategies targeting this specific disease remain limited. Here, we identify TTLL4, a mono-glutamate glutamyltransferase, as a selective vulnerability in NPM1c AML. TTLL4 catalyzes post-translational hyper-glutamylation of NPM1c at E126, stabilizes its cytoplasmic localization and promotes a differentiation block in leukemic cells. Multiple genetic TTLL4 inactivation approaches in human NPM1c-mutant cell lines reduce NPM1c glutamylation, trigger myeloid differentiation, and impair proliferation. Transcriptomic analyses show that TTLL4 knockdown pheno-copies NPM1c degradation and aligns with KMT2A and XPO1-targeted gene expression programs. Furthermore, Ttll4 knockout significantly prolonged survival in an NPM1c/NRAS-driven mouse AML model and promoted differentiation. We identify a small molecule, EN7, that selectively inhibits TTLL4 and recapitulates these phenotypes in NPM1c+ cells. These findings identify glutamylation as a new axis of leukemic regulation and highlight TTLL4 as a druggable epigenetic regulator in NPM1c AML.

cancer biology↗

bioRxiv2022

Splicing modulators impair DNA damage response and induce killing of cohesin-mutant MDS/AML

Splicing modulation is a promising treatment strategy pursued to date only in splicing-factor mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in cancer, including myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. Here, we show that cohesin mutations are biomarkers of sensitivity to drugs targeting the splicing-factor SF3B1 (H3B-8800 and E-7107). We identify drug-induced alterations in splicing and corresponding reduced gene expression of a large number of DNA repair genes, including BRCA1 and BRCA2, as the mechanism underlying this sensitivity in cell line models, primary patient samples and patient-derived xenograft (PDX) models of AML. We find that DNA damage repair genes are particularly sensitive to exon skipping induced by SF3B1 modulators given their long length and large number of exons per transcript. Furthermore, we demonstrate that treatment of cohesin-mutant cells with SF3B1 modulators not only results in impaired DNA damage response and accumulation of DNA damage, but it significantly sensitizes cells to subsequent killing by PARP inhibitors and chemotherapy, and leads to improved overall survival of PDX models of cohesin-mutant AML in vivo. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS and AML, and we propose this as a broader strategy for therapeutic targeting of other DNA damage-repair deficient cancers. One Sentence SummaryWe identify an unexpected effect of SF3B1 splicing inhibitors on regulation of DNA damage repair genes and show efficacy of combination treatment in cohesin-mutant MDS and AML.

cancer biology↗

bioRxiv2020

CBL mutations promote activation of PI3K/AKT signaling via LYN kinase

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations occur in myeloid malignancies, but the mechanism by which these mutations drive oncogenesis remains incompletely understood. Here we performed a series of studies to define the phosphoproteome, CBL interactome and molecular mechanisms of signaling activation in cells expressing an allelic series of CBL mutants. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced PIK3R1 recruitment and downstream PI3K/AKT signaling in CBL-mutant cells. Furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary chronic myelomonocytic leukemia cells. Overall, our data provide rationale for exploring the therapeutic potential of LYN inhibition in patients with CBL-mutated myeloid malignancies. Statement of SignificanceWe investigated the oncogenic mechanisms of myeloid malignancy-associated CBL mutations by mass spectrometry-based proteomics and interactomics. Our findings indicate that increased LYN kinase activity in CBL-mutant cells stimulates PI3K/AKT signaling, revealing opportunities for the use of targeted inhibitors in CBL-mutated myeloid malignancies.

cancer biology↗