Biology subjects

Law, B. K.

Publications and source records attributed to Law, B. K..

10 recordsLinked to original sources

bioRxiv2025

Mapping embryonic mouse lung development using enhanced spatial transcriptomics

The mature mammalian lung contains [~]50 different cell types that are located in precise positions along and around the airway epithelial tree. Here, we investigated how these spatial patterns are progressively established during development. Specifically, we optimized a microfluidics-enabled, deterministic barcoding-based sequencing (DBiT-seq) workflow to achieve highly sensitive spatial mapping of tissue sections from early embryonic mouse lungs. Spatial mapping of epithelial populations revealed that Sox9+ epithelial progenitors are located throughout the epithelial tree at early stages before becoming confined to the distal ends. Spatial mapping of mesenchymal populations revealed a previously unrecognized mesenchymal cluster that expresses high levels of extracellular matrix proteins and appears to promote embryonic lung innervation. These findings provide new insights into the spatial organization and cellular dynamics underlying early lung development, and demonstrate the power of spatial transcriptomics to uncover hidden patterns and populations of cells.

developmental biology↗

bioRxiv2025

CerS2 is a druggable target in triple-negative breast cancer

Triple-negative breast cancer (TNBC) remains a significant clinical challenge due to its aggressive nature and lack of effective targeted therapies. The enzyme ceramide synthase 2 (CerS2), which synthesizes pro-apoptotic very long-chain ceramides (VLCCs), represents a promising therapeutic target. Here, we identify and characterize DH20931, a novel, first-in-class small-molecule agonist of CerS2. We demonstrate that DH20931 directly activates CerS2 with nanomolar potency, leading to significant VLCC accumulation in breast cancer cells. This lipotoxic event induces endoplasmic reticulum (ER) stress and triggers apoptosis via the canonical ATF4/CHOP/PUMA signaling pathway. Mechanistically, we uncover a novel interaction between CerS2 and the ER calcium channel, Inositol 1,4,5-trisphosphate receptor 1 (IP3R1). We demonstrate that DH20931 promotes this interaction, enhancing ER-mitochondria proximity and facilitating a CerS2-dependent flux of calcium (Ca{superscript 2}) from the ER into mitochondria. This subsequent mitochondrial Ca{superscript 2} overload serves as a critical trigger for apoptosis. In preclinical evaluations, DH20931 potently inhibited the growth of TNBC cells in 2D and 3D cultures and significantly suppressed tumor progression in orthotopic and patient-derived xenograft (PDX) models, all while exhibiting a favorable safety profile. Our findings validate CerS2 as a druggable target in TNBC and establish a novel therapeutic strategy that leverages a coordinated attack on cancer cells through ER stress and calcium-mediated mitochondrial dysfunction. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/670525v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@2db377org.highwire.dtl.DTLVardef@6175d4org.highwire.dtl.DTLVardef@7edb8corg.highwire.dtl.DTLVardef@80042e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDH20931 is a first-in-class, potent agonist of Ceramide Synthase 2 (CerS2). C_LIO_LICerS2 activation induces ER stress and engages the ATF4/CHOP/PUMA apoptotic pathway. C_LIO_LIDH20931 promotes a novel CerS2-IP3R1 interaction, causing lethal mitochondrial calcium overload. C_LIO_LITargeting CerS2 shows significant preclinical efficacy against triple-negative breast cancer. C_LI

cancer biology↗

bioRxiv2025

Orthogonal resistance mechanisms of classical- and induced-proximity inhibitors

Resistance development is an inevitable failure mode of many drugs, pointing to the need to develop agents with orthogonal resistance mechanisms. Induced-proximity modalities, an emergent class of therapeutics, operate by forming a ternary complex with the protein-of-interest (POI) and effectors, unlike classical inhibitors that form binary complexes with the POI. Using KRAS as a model system, we employed base editor tiling mutagenesis screening to show that induced-proximity inhibitors exhibit orthogonal resistance mechanisms to classical inhibitors despite overlapping binding sites, offering an opportunity to circumvent resistance mechanisms of classical inhibitors. These findings highlight the use of base editor mutagenesis screens to prioritize inhibitors with orthogonal resistance mechanisms and the potential of induced-proximity inhibitors to overcome the drug resistance of classical inhibitors.

biochemistry↗

bioRxiv2024

DR5 disulfide bonding as a sensor and effector of protein folding stress

New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.

cancer biology↗

bioRxiv2023

A molecular glue approach to control the half-life of CRISPR-based technologies

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latters rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

bioengineering↗

bioRxiv2021

Repurposing Tranexamic Acid as an Anticancer Agent

Tranexamic Acid (TA) is a clinically used antifibrinolytic that acts as a lysine mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic lysine and arginine, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

cancer biology↗

bioRxiv2021

Sensitization of FOLFOX-resistant colorectal cancer cells via the modulation of a novel pathway involving protein phosphatase 2A

The treatment of colorectal cancer (CRC) with FOLFOX shows some efficacy, but these tumors quickly develop resistance to this treatment. We have observed an increased phosphorylation of AKT1/mTOR/4EBP1 and levels of p21 in FOLFOX-resistant CRC cells. We have identified a small molecule, NSC49L, that stimulates protein phosphatase 2A (PP2A) activity, downregulates the AKT1/mTOR/4EBP1-axis, and inhibits p21 translation. We have provided evidence that NSC49L- and TRAIL-mediated sensitization is synergistically induced in p21-knockdown CRC cells, which is reversed in p21-overexpressing cells. p21 binds with procaspase 3 and prevents activation of caspase 3. We have shown that TRAIL induces apoptosis through the activation of caspase 3 by NSC49L-mediated downregulation of p21 translation, and thereby cleavage of procaspase 3 into caspase 3. NSC49L does not affect global protein synthesis. These studies provide a mechanistic understanding of NSC49L as a PP2A agonist, and how its combination with TRAIL sensitizes FOLFOX-resistant CRC cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/453259v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@bc3332org.highwire.dtl.DTLVardef@1c533b0org.highwire.dtl.DTLVardef@1c4a852org.highwire.dtl.DTLVardef@6aec2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

cancer biology↗

bioRxiv2021

Identification of the Molecular Targets of Disulfide Bond Disrupting Agents

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

pharmacology and toxicology↗

bioRxiv2019

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic {beta} cells to secrete protective immunomodulatory factor interleukin-10.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC=\"FIGDIR/small/732354v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (30K):\norg.highwire.dtl.DTLVardef@48a34aorg.highwire.dtl.DTLVardef@d886d0org.highwire.dtl.DTLVardef@17acc61org.highwire.dtl.DTLVardef@fffe44_HPS_FORMAT_FIGEXP M_FIG TOC GRAPHIC C_FIG

synthetic biology↗