Biology subjects

Slabicki, M.

Publications and source records attributed to Slabicki, M..

9 recordsLinked to original sources

bioRxiv2026

A Lenalidomide-Inducible Suicide Switch for Gene- and Cell-Based Therapy

Genetically modified immune effector cells, including chimeric antigen receptor (CAR) T cells, have emerged as transformative anti-cancer agents. However, these autonomous, living therapies are limited by difficult-to-control hyperactivation toxicities. To achieve their full potential as therapies for a broader range of diseases, CAR T cells must target novel antigens and integrate further potency enhancements that raise the possibility of novel and intensified toxicities. High performance safety systems are needed to restrain these highly active next-generation gene- and cell-based therapies. Cell therapy suicide switches remain infrequently used, and are currently limited by inaccessible controller drugs, incomplete cell depletion, fitness cost, and immunogenicity. To address these issues, we engineered a fully human sequence-based cell therapy suicide switch based on the overexpression of Caspase-Activated DNase (CAD) and its stoichiometric chaperone/inhibitor ICAD fused to a lenalidomide-inducible degron. Acute destabilization of ICAD with the broadly available, off-patent molecular glue degrader drug lenalidomide uncaged CAD to induce DNA damage and cell death. By this degradable cage mechanism, the ICAD-CAD suicide switch achieved long-term expression in primary human T cells without detectable leakiness, and deep drug-induced cell depletion. The ICAD-CAD switch was generalizable to alternative acute destabilization systems. Genome-wide CRISPR screening established the necessity of CAD and the lenalidomide-interacting E3 ubiquitin ligase substrate adaptor CRBN for switch function. When co-delivered with a CAR, ICAD-CAD CAR T cells retained effector functions and could be rapidly depleted with lenalidomide.

synthetic biology↗

bioRxiv2026

Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

PPM1D is a serine/threonine phosphatase and DNA damage response (DDR) regulator recurrently activated in cancer through amplification or C-terminal truncating mutations that increase its abundance. Here we show that truncating mutations fundamentally rewire PPM1D proteostasis, unmasking an oncogenic function of an alternative protein degradation pathway. While full-length PPM1D undergoes rapid ubiquitin-independent proteasomal degradation via a C-terminal degron, truncating mutations redirect degradation to a slower UBR5-mediated ubiquitin-dependent pathway. The resulting accumulation of PPM1D suppresses DDR signaling and enhances cellular fitness under genotoxic stress, which is further amplified by UBR5 loss. Consistent with selective pressure on this axis, cancers harboring PPM1D truncating mutations are enriched for UBR5 loss-of-function mutations. Together, these findings identify escape from ubiquitin-independent proteasomal degradation as a mechanism of oncogenic adaptation and establish proteostatic routing as a regulatory layer linking protein degradation, DDR signaling, and cancer evolution.

cell biology↗

bioRxiv2025

Discovery of chromatin-based determinants of azacytidine and decitabine anti-cancer activity

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved - specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of USP48, which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition in vitro and in vivo. Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

cancer biology↗

bioRxiv2025

DNMT3A Stability Is Maintained by Ubiquitin-Specific Peptidase 11 (USP11) and Sumoylation Countering Degradation

DNA methyltransferase 3A (DNMT3A) plays crucial roles in mammalian development and hematopoiesis. DNMT3A protein instability is associated with blood diseases such as myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), as well as Tatton-Brown-Rahman syndrome, an overgrowth disorder. We found that certain unstable DNMT3A mutations cause DNMT3A localization changes, resulting in loss of function. This mislocalization is partially rescued by E1 enzyme inhibition or stable USP11 expression, as DNMT3A stability is maintained by deubiquitinating enzyme USP11 countering degradation by CUL4-DCAF8 E3 ligase. We also found that USP11 enhances DNMT3A SUMOylation by promoting the interaction between DNMT3A and SUMO E3 ligases. DNMT3A SUMOylation also is essential to maintain DNMT3A protein stability. Furthermore, we found USP11 enhances the binding of DNMT3A to the polycomb complex and maintains DNMT3A DNA methyltransferase (MTase) activity. This study uncovers the mechanism for DNMT3A protein turnover through USP11, which is essential to DNMT3A function, and may be a therapeutic approach for diseases caused by DNMT3A protein instability.

biochemistry↗

bioRxiv2024

Discovery of electrophilic degraders that exploit SNAr chemistry

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly undruggable targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

biochemistry↗

bioRxiv2024

Charged Molecular Glue Discovery Enabled by Targeted Degron Display

Small molecules that induce protein interactions hold tremendous potential as new medicines, as probes for molecular pathways, and as tools for agriculture. Explosive growth of targeted protein degradation (TPD) drug development has spurred renewed interest in proximity-inducing molecules and especially Molecular Glue Degraders (MGDs). These compounds catalyze destruction of disease-causing proteins by reshaping protein surfaces and promoting cooperative binding between ubiquitylating enzymes and target proteins. MGD discovery for pre-defined targets is a major challenge in contemporary drug discovery. The field is limited by a lack of approaches that can exploit charged ligand-binding pockets, thus excluding a major fraction of ubiquitin ligases (E3s) that evolved to recognize exceedingly common acidic and basic degrons. Here we solve these important chemical challenges through "chemocentric" MGD discovery of ZZ1, a BET-family protein degrader and a prodrug of a negatively charged glue (c-Glue). ZZ1 activation unmasks a sulfinic acid moiety that binds the modular GID/CTLH ubiquitin ligase complex via a basic pocket in its YPEL5 subunit. YPEL5 is a CRBN structural homolog and an essential non-Cullin ubiquitin ligase cofactor expressed in cancers of the bone marrow. These findings demonstrate a previously unrecognized capacity of YPEL5 to recruit GID/CTLH substrates, and they provide a powerful strategy to discover c-Glues that induce proximity to ubiquitin ligases with similarly desirable properties.

biochemistry↗

bioRxiv2023

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 [A] cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

biochemistry↗

bioRxiv2020

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using a Compressed Barcode Space

The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day.

molecular biology↗

bioRxiv2018

MDM4 is an essential disease driver targeted by 1q gain in Burkitt lymphoma

Oncogenic MYC activation promotes cellular proliferation in Burkitt lymphoma (BL), but also induces cell cycle arrest and apoptosis mediated by TP53, a tumor suppressor gene that is mutated in 40% of BL cases. To identify therapeutic targets in BL, we investigated molecular dependencies in BL cell lines using RNAi-based, loss-of-function screening. By integrating genotypic and RNAi data, we identified a number of genotype-specific dependencies including the dependence of TCF3/ID3 mutant cell lines on TCF3 and of MYD88 mutant cell lines on TLR signaling. TP53 wild-type (TP53wt) BL were dependent on MDM4, a negative regulator of TP53. In BL cell lines, MDM4 knockdown induced cell cycle arrest and decreased tumor growth in a xenograft model in a p53-dependent manner, while small molecule inhibition of the MDM4-p53 interaction restored p53 activity resulting in cell cycle arrest. Consistent with the pathogenic effect of MDM4 upregulation in BL, we found that TP53wt BL samples were enriched for gain of chromosome 1q which includes the MDM4 locus. 1q gain was also enriched across non-BL cancer cell lines (n=789) without TP53 mutation (23% in TP53wt and 12% in TP53mut, p<0.001). In a set of 216 cell lines representing 19 cancer entities from the Achilles project, MDM4 was the strongest genetic dependency in TP53wt cell lines (p<0.001).\n\nOur findings show that in TP53wt BL, MDM4-mediated inhibition of p53 is a mechanism to evade cell cycle arrest. The data highlight the critical role of p53 as a tumor suppressor in BL, and identifies MDM4 as a key functional target of 1q gain in a wide range of cancers, which is therapeutically targetable.

cancer biology↗