Biology subjects

Kwok, H. S.

Publications and source records attributed to Kwok, H. S..

9 recordsLinked to original sources

bioRxiv2026

Pathway-wide base editing charts chemical-genetic interactions in MAPK signaling

CRISPR base editor (BE) scanning enables sequence-level interrogation of proteins at scale in their native cellular and genomic contexts. This approach opens new opportunities to dissect cellular pathways, where signaling depends on coordinated interactions among multiple pathway proteins. We apply BE scanning to the RAS-RAF-MEK-ERK (MAPK) cascade, a central oncogenic pathway and major therapeutic target. Compounds targeting the MAPK pathway have mechanisms of action and resistance that remain incompletely characterized. By base editing 22 MAPK genes under either hyperactivation or inhibition at various nodes, we present a chemical-genetic map of the signaling pathway. Pathway-wide analysis shows that drug resistance mutations frequently occur in proteins other than the direct drug target itself, and we exploit these chemical-genetic interactions to recapitulate key pathway connections. Our BE scanning also identified underappreciated functional hotspots, including an allosteric site in the KRAS N-terminus. We also identify catalytically impaired CRAF mutants that confer bidirectional resistance and sensitization to structurally similar MEK inhibitors, distinguishing functional differences between structurally related compounds. These findings establish BE scanning as a scalable approach for network-level chemical-genetic interaction mapping, revealing new mechanistic insights into one of the most studied oncogenic pathways.

cancer biology↗

bioRxiv2025

A chemical-genetic interaction between PAF1 and ENL/AF9 YEATS inhibition

Transcriptional regulatory proteins are frequent drivers of oncogenesis and common targets for drug discovery. The transcriptional co-activator, ENL, which is localized to chromatin through its acetyllysine-binding YEATS domain, is preferentially required for the survival and pathogenesis of acute leukemia. Small molecules that inhibit the ENL YEATS domain show anti-leukemia effects in preclinical models, which is thought to be caused by the downregulation of pro-leukemic ENL target genes. However, the transcriptional effects of ENL YEATS domain inhibitors have not been studied in models of intrinsic or acquired resistance and, therefore, the connection between proximal transcriptional effects and downstream anti-proliferative response is poorly understood. To address this, we identified models of intrinsic and acquired resistance and used them to study the effects of ENL YEATS domain inhibitors. We first discovered that ENL YEATS domain inhibition produces similar transcriptional responses in naive models of sensitive and resistant leukemia. We then performed a CRISPR/Cas9-based genetic modifier screen and identified in-frame deletions of the essential transcriptional regulator, PAF1, that confer resistance to ENL YEATS domain inhibitors. Using these drug-resistance alleles of PAF1 to construct isogenic models, we again found that the downregulation of ENL target genes is shared in both sensitive and resistant leukemia. Altogether, these data support the conclusion that the suppression of ENL target genes is not sufficient to explain the anti-leukemia effects of ENL antagonists.

biochemistry↗

bioRxiv2025

Phosphorylation-inducing chimera rewires oncogenic kinase to trigger apoptosis

The active sites electric field is integral to enzymatic catalysis (e.g., substrate recognition) and nature employs charge-altering post-translational modifications (e.g., phosphorylation) to perturb this electric field and regulate enzymes. A chromosomal translocation converts Abelson kinase (ABL) to BCR-ABL, whose hyperactivity drives several cancers. Here, we developed a small molecule, BRD8833, that induces BCR-ABL phosphorylation, which perturbs its active sites electric field with loss of hyperactivity. Unlike "occupancy-driven" inhibitors that require stoichiometric concentrations, BRD8833 operates through an event-driven, substoichiometric mechanism by inducing proximity between two BCR-ABL molecules to trigger the inhibitory phosphorylation and selective apoptosis of BCR-ABL-dependent cancer cells. Furthermore, BRD8833 is effective against other oncogenic ABL fusions or clinically observed resistance mutations, including those to occupancy-driven drugs with the same binding site as BRD8833, suggesting differences in their resistance mechanisms. These studies lay the foundation for electric-field and "event-driven" modalities to control hyperactive enzymes with orthogonal resistance mechanisms to occupancy-driven drugs. GRAPHICAL ABSTRACT (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/659082v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1607a40org.highwire.dtl.DTLVardef@fa10deorg.highwire.dtl.DTLVardef@1698232org.highwire.dtl.DTLVardef@1e2e0f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Coupling CRISPR Scanning with Targeted Chromatin Accessibility Profiling using a Double-Stranded DNA Deaminase

Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms and the development of gene therapies. However, these approaches cannot be combined with direct scalable readouts of chromatin structure and accessibility across long single-molecule chromatin fibers. Here we leverage a double-stranded DNA cytosine deaminase to profile chromatin accessibility at high depth and resolution at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). Powered by high sequence coverage at targeted loci of interest, TDAC-seq can be uniquely integrated with CRISPR perturbations to enable the functional dissection of cis-regulatory elements, where genetic perturbations and their effects on chromatin accessibility are superimposed on the same single chromatin fiber and resolved at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34+ hematopoietic stem and progenitor cells during erythroid differentiation as well as in pooled CRISPR and base editing screens tiling an enhancer controlling the globin locus. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.

molecular biology↗

bioRxiv2024

Asymmetric Engagement of Dimeric CRL3KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal1, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

molecular biology↗

bioRxiv2024

KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function1. As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB)2, the most common embryonal brain tumor in children, and pineoblastoma3. These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST4. However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.

molecular biology↗

bioRxiv2022

Drug addiction mutations unveil a methylation ceiling in EZH2-mutant lymphoma

Cancer mutations in Polycomb Repressive Complex 2 (PRC2) drive aberrant epigenetic states. Although therapies inhibiting the PRC2 enzymatic component EZH2 are FDA-approved, oncogene-specific dependencies remain to be discovered. Here, we identify mutations that confer both resistance and drug addiction to PRC2 inhibitors in EZH2-mutant lymphoma, resulting in cancer cells that paradoxically depend on drug for survival. Drug addiction is mediated by hypermorphic mutations in the CXC domain of EZH2, which maintain H3K27me3 levels even in the presence of PRC2 inhibitors. Drug removal leads to overspreading of H3K27me3, surpassing a repressive methylation ceiling compatible with lymphoma cell survival. Activating EZH2 cancer mutations establish an epigenetic state precariously close to this ceiling, which we show can be breached by inhibition of SETD2, a PRC2 antagonist, to block lymphoma growth. More broadly, we highlight how approaches to identify drug addiction mutations can be leveraged to discover cancer vulnerabilities.

cancer biology↗

bioRxiv2020

Exploiting evolutionary trade-offs to combat antibiotic resistance

Antibiotic resistance frequently evolves through fitness trade-offs in which the genetic alterations that confer resistance to a drug can also cause growth defects in resistant cells. Here, through experimental evolution in a microfluidics-based turbidostat, we demonstrate that antibiotic-resistant cells can be efficiently inhibited by amplifying the fitness costs associated with drug-resistance evolution. Using tavaborole-resistant E. coli as a model, we show that genetic mutations in leucyl-tRNA synthetase (that underlie tavaborole resistance) make resistant cells intolerant to norvaline, a chemical analog of leucine that is mistakenly used by tavaborole-resistant cells for protein synthesis. We then show that tavaborole-sensitive cells quickly outcompete tavaborole-resistant cells in the presence of norvaline due to the amplified cost of the molecular defect of tavaborole resistance. This finding illustrates a potentially generalizable approach for combating therapeutic resistance, prolonging the effectiveness of drugs and enabling the use of drugs that are no longer effective due to the rapid evolution of resistance.

evolutionary biology↗

bioRxiv2019

Archaeal ribosomal proteins possess nuclear localization signal-type motifs: implications for the origin of the cell nucleus

AO_SCPLOWBSTRACTC_SCPLOWEukaryotic cells are divided into the nucleus and the cytosol, and, to enter the nucleus, proteins typically possess short signal sequences, known as nuclear localization signals (NLSs). Although NLSs have long been considered as features unique to eukaryotic proteins, we show here that similar or identical protein segments are present in ribosomal proteins from the Archaea. Specifically, the ribosomal proteins uL3, uL15, uL18, and uS12 possess NLS-type motifs that are conserved across all major branches of the Archaea, including the most ancient groups Microarchaeota and Diapherotrites, pointing to the ancient origin of NLS-type motifs in the Archaea. Furthermore, by using fluorescence microscopy, we show that the archaeal NLS-type motifs can functionally substitute eukaryotic NLSs and direct the transport of ribosomal proteins into the nuclei of human cells. Collectively, these findings illustrate that the origin of NLSs preceded the origin of the cell nucleus, suggesting that the initial function of NLSs was not related to intracellular trafficking. Overall, our study reveals rare evolutionary intermediates among archaeal cells that can help elucidate the sequence of events that led to the origin of the eukaryotic cell.

evolutionary biology↗