Biology subjects

Chang, W.-H.

Publications and source records attributed to Chang, W.-H..

12 recordsLinked to original sources

bioRxiv2026

KRASG12C Inhibition Remodels the Non-Small Cell Lung Cancer Surfaceome to Uncover Targetable Vulnerabilities

Mutant-selective and pan-RAS(ON) multi-selective inhibitors have improved clinical outcomes for patients with RAS-driven cancers; however, adaptive resistance limits the depth and durability of response. Using an integrated genetic, transcriptomic, and mass-spectrometry-based quantitative temporal proteomic platform, we profile the adaptive response to KRASG12C inhibitors (KRASi) in KRASG12C-mutant lung adenocarcinoma models. Our analyses reveal remodeling of signaling pathways and the cell surface proteome, including cell surface proteins selectively upregulated across cell lines, tumor models, and treatment durations. Folate receptor alpha (FOLR1) emerged as a therapeutically actionable target that is significantly upregulated with KRASi and is part of a broader acute cellular state transition characterized by acquisition of an epithelial identity. Combining KRASi with mirvetuximab soravtansine-gynx, a clinically approved FOLR1-targeting antibody drug conjugate, enhanced tumor cell cytotoxicity and deepened response and tumor control in cellular and in vivo models. Additionally, this work represents a comprehensive multi-omic resource characterizing non-genetic temporal adaptation to KRASi for future mechanistic and therapeutic investigations.

cancer biology↗

bioRxiv2026

An On-Demand Nanodisc Platform for Reconstitution of Functional Membrane Proteins into Model and Living Membranes

Membrane proteins are central to transport, signaling, and pharmacological regulation, yet their direct functional reconstitution into defined membrane environments remains technically challenging. Detergent-based workflows have enabled major advances in membrane protein research, but some applications require complementary strategies that better preserve native-like lipid environments. Cell-based expression approaches, meanwhile, require long incubation times and suffer from cell-type-dependent variability. Here, we establish nanodiscs as modular carriers for the rapid delivery of both lipids and full-length membrane proteins into model and cellular membranes. Using supported lipid bilayers, we first show that membrane scaffold protein (MSP) nanodiscs mediate efficient lipid transfer within minutes, with fluorescence recovery after photobleaching confirming lateral mobility of the delivered lipids. We then extend this strategy to the bacterial calcium channel BsYetJ, achieving concentration-dependent protein incorporation and single-molecule diffusion within supported lipid bilayers. Importantly, BsYetJ-loaded nanodiscs enable direct reconstitution of functional channels into intact mammalian plasma membranes across multiple cell lines. Calcium imaging demonstrates robust BsYetJ-mediated calcium influx, confirming that the delivered channel retains ion-conductive activity after transfer into heterologous cellular membranes. Crucially, this nanodisc-mediated delivery bypasses the variable trafficking pathways inherent to different host systems, allowing for the direct reconstitution of membrane proteins into target membranes while preserving their functional activity. Furthermore, unlike MSP nanodiscs, styrene-maleic acid (SMA) nanodiscs can directly capture membrane proteins from native cell membranes. This capability makes them particularly well-suited for studying complex and challenging membrane proteins. Therefore, we further generalize this platform using SMA nanodiscs . We demonstrate that, similar to MSP nanodiscs, SMA nanodiscs can efficiently deliver lipid cargo to supported bilayers and mammalian cells. By directly capturing full-length dopamine D2 receptor from cellular membranes and transferring it into naive target cells, we achieve functional GPCR reconstitution, as validated by specific binding of a custom fluorescent agonist. Together, these results demonstrate that nanodiscs can serve not only as stabilizing membrane mimetics but also as active delivery vehicles for on-demand membrane protein reconstitution. This approach provides a rapid and broadly applicable platform for interrogating ion channels, GPCRs, and other challenging pharmacological targets in user-defined membrane environments.

biochemistry↗

bioRxiv2026

Concurrent AXL inhibition enhances RAS and ERK inhibitor efficacy in KRAS-mutant pancreatic and lung cancer

Resistance limits the clinical efficacy of RAS inhibitors. We applied chemical and genetic screens and identified the AXL receptor tyrosine kinase as a driver of resistance to RAS-ERK inhibition. We determined that combination treatment with the AXL inhibitor bemcentinib (AXLi) together with the RAS(ON) multi-selective tri-complex inhibitor RMC-7977 (RASi) or the ERK-selective inhibitor SCH772984 (ERKi) significantly enhanced growth suppression in human KRAS-mutant pancreatic and lung cancer models. Combined AXLi and RASi treatment of human KRAS-mutant pancreatic cell line-derived xenograft tumors synergistically suppressed ERK activation and MYC expression, and caused tumor regression. Analyses of immunocompetent mouse allograft pancreatic tumor models revealed a largely tumor cell-intrinsic response to inhibitor treatment. We identified an unexpected mechanism whereby KRAS inhibition upregulated the AXL ligand GAS6, activating AXL but inducing an AXL-dependent adaptive resistance mechanism wherein AXL antagonizes RASi efficacy. Our observations support concurrent AXL inhibition as a strategy to enhance RAS inhibitor clinical efficacy. STATEMENT OF SIGNIFICANCEOur findings identify AXL as a driver of resistance to RAS inhibitors, establishing a combination strategy to overcome resistance and enhance RAS inhibitor therapeutic efficacy in KRAS-mutant cancer by maximally inhibiting oncogenic RAS signaling.

cancer biology↗

bioRxiv2026

Characterization and therapeutic suppression of KEAP1-NRF2-driven resistance to KRAS inhibitors in pancreatic and lung cancer

The recent approval of KRAS inhibitors supports the therapeutic value of targeting mutant KRAS cancers. However, clinical efficacy is hindered by both primary and treatment-associated acquired resistance. We applied a CRISPR-Cas9 loss-of-function screen and identified loss of KEAP1 as a resistance mechanism to the KRASG12D-selective inhibitor MRTX1133 and the RAS(ON) multi-selective inhibitor RMC-7977 in pancreatic cancer models. RNA-sequencing analyses revealed a KEAP1KO transcriptome that is distinct from the ERK-, MYC-, and YAP/TAZ-TEAD-dependent transcriptional programs that drive KRAS inhibitor resistance, demonstrating a distinct mechanism of resistance. We then established a PDAC KEAP1-deficient (PKD) gene signature that was enriched in patients and preclinical models insensitive to KRAS inhibitor treatment. Finally, we observed that KEAP1-deficient cells exhibited elevated glutamine metabolism, and combination treatment with the glutamine antagonist DRP-104 (sirpiglenastat) enhanced KRAS inhibitor suppression of pancreatic and lung tumors. SIGNIFICANCEKEAP1 loss is associated with reduced response to KRAS inhibitor therapy. We demonstrate that KEAP1 loss-associated resistance can be overcome by pharmacologic inhibition of the KEAP1 loss-induced glutamine dependency, establishing a combination to enhance RAS inhibitor clinical efficacy.

cancer biology↗

bioRxiv2025

Dordaviprone/ONC201 Activation of the ClpP Mitochondrial Protease Inhibits the Growth of KRAS-Mutant Pancreatic Cancer and Overcomes RAS Inhibitor Resistance

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS-driven oncogenic signaling and tumor growth. Blockade of the KRAS ERK-MAPK pathway via small molecule direct RAS inhibitors has shown clinical promise, but intrinsic and acquired resistance limit the efficacy of these inhibitors as single agents. To identify potential combination strategies, we first assessed the ability of dordaviprone/ONC201, an FDA-approved agent, to inhibit PDAC cell and organoid growth. We observed that ONC201 reduced the growth of a broad panel of KRAS-mutant PDAC cell lines, and that the expression of mitochondrial protease ClpP was required for this efficacy. Mechanistically, we observed that treatment with ONC201 led to inhibition of mitochondrial respiration, causing a compensatory increase in glycolysis. Furthermore, ONC201 caused ClpP-dependent activation of PI3K-AKT-mTOR signaling and concurrent PI3K and mTOR inhibition further enhanced ONC201 growth suppression. ONC201 demonstrated an additive effect when combined with a RAS(ON) multi-selective inhibitor RMC-7977 in PDAC cells and organoids. Finally, PDAC cell lines with acquired resistance to RMC-7977 or KEAP1 loss-driven resistance retained sensitivity to ONC201. We propose that concurrent treatment with ONC201 may delay onset of resistance to RAS inhibitor therapy. Statement of SignificanceClpP activation by dordaviprone/ONC201 suppressed PDAC cell growth and overcame resistance to the RAS(ON) multi-selective inhibitor RMC-7977, providing support for investigating this combination as a potential combination treatment for KRAS-mutant pancreatic cancer.

cancer biology↗

bioRxiv2025

A topologically conserved unstructured region helps positioning the evolutionarily conserved Prp40 WW domains to promote non-canonical intron splicing

Newly transcribed introns are immediately identified by early splicing factors that recognize the intron 5 splice site (5SS) and branch site (BS). In the budding yeast, these critical splice-site sequences are generally constrained, whereas degeneracy is the rule in higher eukaryotes. Yet, [~]40% of the yeast introns do diverge, to a certain degree, from the canonical sequences. Exactly how these non-canonical introns are recognized and spliced remains unknown. Here we show that the conserved Prp40 WW domains promote non-canonical intron splicing by enhancing stable U1 snRNP and BBP recruitments. AlphaFold predicts a topologically conserved unstructured region between Prp40 WW and FF domains. Alignment of the AlphaFold Prp40 structure with published U1 snRNP structure positions WW domains adjacent to 5SS and Luc7, which is known to be critical for 5SS recognition. Indeed, deletion of this unstructured region negatively impacts on splicing of the non-canonical 5SS introns. Taken together, our results suggest that the conserved WW domains may have evolved to deal with the highly degenerate 5SS and BS sequences in higher eukaryotes, so as to accommodate increased splicing complexity. HighlightsO_LIThe evolutionarily conserved Prp40 WW domains promote splicing of introns harboring non-canonical 5 splice site or branch site. C_LIO_LIPrp40 WW domains enhance stable U1 snRNP and BBP recruitments to nascent transcripts containing non-canonical splice sites. C_LIO_LIA topologically conserved unstructured region between WW and FF domains helps to position Prp40 WW domains close to the 5 splice site. C_LIO_LIThe N-terminal WW domain sterically hinders conformational rearrangements required for efficient release of a BBP variant during spliceosome assembly. C_LIO_LIA reporter assay identified 13 non-canonical introns whose splicing, under various environmental conditions, depend on Prp40 WW domains. C_LI

biochemistry↗

bioRxiv2025

A Hierarchical Robust Linear Model for Cryo-EM Map Analysis

Cryo-electron microscopy (cryo-EM) has become a central tool for determining the atomic structures of biological macromolecules, producing three-dimensional reconstruction maps that guide atomic model building. Current quantitative analyses of cryo-EM maps largely rely on simplified Gaussian signal models with fixed width parameters, limiting their ability to capture atom-specific signal characteristics directly from the maps. We propose a robust hierarchical linear (RHL) model for the statistical analysis of paired cryo-EM maps and atomic structures deposited in the EMDB and PDB. Within this framework, the logarithms of local voxel intensities surrounding each atom are modeled using a linearized Gaussian form, and atom-specific amplitude and width parameters are estimated through a hierarchical structure that pools information across atoms of the same type. The primary objective is to provide a statistically grounded framework for extracting quantitative atomic signal features from cryo-EM maps without imposing rigid physical assumptions on these parameters. To address contamination arising from overlapping atomic signals, spatially varying resolution, and experimental noise, we incorporate a data-adaptive weighting scheme based on minimum density power divergence estimation (MDPDE). This formulation preserves computational efficiency and ensures stable parameter estimation, while naturally facilitating the identification of deviated atomic profiles that may stem from model misspecification or local structural heterogeneity. Simulation studies demonstrate that the proposed method yields stable group-level parameter estimates even under substantial contamination. Applications to cryo-EM maps at multiple resolutions show that an atomic resolution (1.25 Angstrom) apoferritin map exhibits systematic atom-type differences in Gaussian amplitude and width, whereas a near-atomic-resolution map (2.20 Angstrom) shows closer agreement with conventional uniform-width assumptions. Beyondstructural biology, the proposed RHL framework provides a general statistical methodology for hierarchical data integration under heterogeneous noise, with potential applications in multi-center studies and federated learning.

bioinformatics↗

bioRxiv2024

Dynamic positioning of a TFIIE-related winged-helix domain in RNA polymerase elongation complex III underpins its multi-functionality

The tandem-winged-helix (tWH) domain of RNA polymerase III (Pol III) Rpc34 subunit is a multi-function hub in Pol III apparatus while the structure basis remains elusive. To probe tWH in Pol III elongation complex (EC), we engineered an azide-bearing unnatural amino acid into tWH and implemented a thiol-capping scheme to enable strain- promoted click reaction for selective dye labeling to suit single-molecule Forster resonance energy transfer (smFRET). Our discrete smFRET results and nano-positioning analysis reveal multiple docking sites of Rpc34 WH2 on Pol III EC with characteristic dwell-times, reflecting promiscuous and weak interactions underpinning tWHs multi-functionality. The docking sites include one overlapping with that during initiation and others on transcription bubble and downstream DNA, previously unreported. This work provides mechanistic insights into Pol III transcription re-initiation and elongation, with useful bio-orthogonal strategies for studying structural dynamics of large native protein complexes.

biophysics↗

bioRxiv2024

Cryo-EM inspired NMR analysis reveals a pH-induced conformational switching mechanism for imparting dynamics to Betanodavirus protrusions

Nervous necrosis virus (NNV), a non-enveloped betanodavirus, causes neuropathies and retinopathies in farmed fish, damaging aquaculture worldwide. NNV has 60 conspicuous surface protrusions comprising the protrusion domain (P-domain) of its capsid protein. Although NNV protrusions play critical roles in infectivity, the underlying dynamics remain unclear. Our cryogenic electron microscopy (cryo-EM)-derived structures of Dragon grouper (Epinephelus lanceolatus) NNV reveal that the protrusions undergo low-pH-induced compaction and movement. We show that the P-domain is monomeric in solution at a pH germane to infection (7.0). Moreover, nuclear magnetic resonance (NMR) structures reveal a peptide (amino acids 311-330) that adopts a flexible loop to form an open pocket. NMR spectral analysis at pH 5.0 aided by molecular dynamics (MD) simulations show that this loop switches to a {beta}-strand under acidic conditions, eliciting pocket closure and P-domain trimerization, highlighting a unique pH-sensing feature. Our docking analysis revealed the N-terminal moiety of sialic acid inserted into and interacting with conserved residues in the pocket. Additionally, a low-pH-induced conformational change in the linker region via peptide bond isomerization conferred malleability on the protrusions. Our work uncovers the protrusion dynamics of a betanodavirus governing its infectivity through a pH-dependent conformational switching mechanism, providing insights into complex virus-host interactions.

biochemistry↗

bioRxiv2023

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 angstrom

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating cancer cell toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.

biochemistry↗

bioRxiv2022

RE2DC: a robust and efficient 2D classifier with visualization for processing massive and heterogeneous cryo-EM data

Single-particle cryo-electron microscopy (cryo-EM) increasingly generates millions of particle images, yet two-dimensional (2D) classification remains a major bottleneck because existing approaches balance computational efficiency against robustness to noise, outliers and structural heterogeneity. We introduce RE2DC (Robust and Efficient 2D Classifier), an algorithmic framework that resolves this trade-off through dynamic linear-time clustering, dimension-reduction multi-reference alignment, and offers real-time interactive t-SNE visualization. Rather than relying primarily on hardware acceleration, RE2DC reduces the computational cost of robust clustering and employs de-noised images for alignment, enabling efficient execution on standard multi-core CPUs. Across diverse benchmark datasets, RE2DC achieves class homogeneity comparable to ISAC while processing datasets three- to ten-fold faster per classification round than RELION. Notably, RE2DC resolves rare, structurally coherent particle populations, enabling detection of transient conformational intermediates and supporting near real-time cryo-EM analysis. By addressing algorithmic complexity, RE2DC establishes a general framework for robust, scalable analysis of massive and heterogeneous image datasets.

bioinformatics↗

bioRxiv2019

Small molecule inhibition of PIKFYVE kinase rescues gain- and loss-of-function C9ORF72 ALS/FTD disease processes in vivo

The most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion (HRE) in C9ORF72 that contributes to neurodegeneration by both loss-of-function (decreased C9ORF72 protein levels) and gain-of-function (e.g. dipeptide repeat protein production) mechanisms. Although therapeutics targeting the gain-of-function mechanisms are in clinical development, it is unclear if these will be efficacious given the contribution of C9ORF72 loss-of-function processes to neurodegeneration. Moreover, there is a lack of therapeutic strategies for C9ORF72 ALS/FTD with demonstrated efficacy in vivo. Here, we show that small molecule inhibition of PIKFYVE kinase rescues both loss- and gain-of-function C9ORF72 disease mechanisms in vivo. We find that the reduction of C9ORF72 in mouse motor neurons leads to a decrease in early endosomes. In contrast, treatment with the PIKFYVE inhibitor apilimod increases the number of endosomes and lysosomes. We show that reduced C9ORF72 levels increases glutamate receptor levels in hippocampal neurons in mice, and that apilimod treatment rescues this excitotoxicity-related phenotype in vivo. Finally, we show that apilimod also alleviates the gain-of-function pathology induced by the C9ORF72 HRE by decreasing levels of dipeptide repeat proteins derived from both sense and antisense C9ORF72 transcripts in hippocampal neurons in vivo. Our data demonstrate the neuroprotective effect of PIKFYVE kinase inhibition in both gain- and loss-of-function murine models of C9ORF72 ALS/FTD.

neuroscience↗