Biology subjects

Kim, S.

Publications and source records attributed to Kim, S..

At least 433 records · Page 24Linked to original sources

bioRxiv2022

Discovery and characterization of a first-in-field transcription factor BRN2 inhibitor for the treatment of neuroendocrine prostate cancer.

The increased incidence of treatment-emergent neuroendocrine prostate cancer (NEPC) is particularly alarming as this diagnosis is associated with poor prognosis. Despite initial responses to platinum-based chemotherapy, relapses are common and there is no effective second line therapy for NEPC. We previously identified that neuronal transcription factor BRN2 (POU3F2) is a potent driver of neuroendocrine differentiation and an attractive target for NEPC. Utilizing a combination of in silico modeling and X-ray crystallography followed by structure-based lead optimization, we have developed the first potent, specific and orally bioavailable BRN2 inhibitor (B18-94), which inhibits the interaction between BRN2 and DNA. This loss of BRN2 on the chromatin drastically reduces its transcriptional output resulting in downregulation of several known targets in NEPC such as SOX2, ASCL1 and PEG10. Additionally, B18-94 reduces specifically cell proliferation specifically in multiple NEPC models with no effect on adenocarcinoma and other BRN2 negative prostate cancer models. Importantly, the consistency in the transcriptomic changes driven by B18-94 and or CRISPR/Cas9 mediated BRN2 knockout confirmed the on-target specificity, with both methods of BRN2 inhibition downregulating pathways involved in cellular plasticity and proliferation. Finally, we have demonstrated that B18-94, the first-in-field POU-domain transcription factor inhibitor, significantly reduced tumor growth in several NEPC xenograft models with no observable toxicity, suggesting potential for therapeutic intervention of NEPC.

cancer biology↗

bioRxiv2022

BMAL1 loss in oligodendroglial lineage cells dysregulates myelination and sleep

Myelination depends on maintenance of oligodendrocytes that arise from oligodendrocyte precursor cells (OPCs). We show that the dynamic nature of oligodendroglia and myelination are regulated by the circadian transcription factor BMAL1. Bmal1 knockdown in OPCs during development - but not adulthood - decreases OPC proliferation, whereas BMAL1 regulates OPC morphology throughout life. OPC-specific Bmal1 deficiency impairs remyelination in an age-dependent manner, suggesting that age-associated decrements in circadian regulation of oligodendroglia may contribute to the deficient remyelination potential in demyelinating diseases like multiple sclerosis (MS). This oligodendroglial dysregulation and dysmyelination increase sleep fragmentation in OPC-specific Bmal1 knockout mice, and sleep fragmentation is causally associated with MS. These findings have broad mechanistic and therapeutic implications for numerous brain disorders that include both myelin and sleep phenotypes. One-Sentence SummaryBMAL1 regulates the homeostatic maintenance of oligodendroglia and myelin, that subsequently controls sleep architecture.

neuroscience↗

bioRxiv2022

Digital data repository and automatic analysis framework for FDOPA PET neuroimaging

IntroductionFDOPA PET has been used extensively to image the human brain in many clinical disorders and has the potential to be used for patient stratification and individualized treatment. However, to reach its full and effective clinical translation, FDOPA PET requires both a robust data infrastructure and analytical protocol that are capable of ensuring high quality data and metadata, accurate biological quantification, and replicable results. In this study we evaluate a digital data repository and automated analytical framework for FDOPA PET neuroimaging that can produce an individualised quantification of dopamine synthesis capacity in the living human brain. MethodsThe imaging platform XNAT was used to store the Kings College London institutional brain FDOPA PET imaging archive, alongside individual demographics and clinical information. A fully automated analysis pipeline for imaging processing and data quantification was developed in Python and integrated in XNAT using the Docker technology. Reproducibility was assessed in test-retest datasets both in controls and patients with psychosis. The agreement between the automated analysis estimates and the results derived by the manual analysis were compared. Finally, using a sample of healthy controls (N=115), a sensitivity analysis was performed to explore the impact of experimental and demographic variables on the FDOPA PET measures. ResultsThe final data repository includes 892 FDOPA PET scans organized from 23 different studies, collected at five different imaging sites. After removing commercials studies, the infrastructure consisted of 792 FDOPA PET scans from 666 individuals (female 33.9%, healthy controls 29.1%) collected from four different imaging sites between 2004-2021. The automated analysis pipeline provided results that were in agreement with the results from the manual analysis, with a Pearsons correlation that ranged from 0.64 to 0.99 for Kicer, and from 0.79 to 1.00 for SUVR. The mean absolute difference between the two pipelines ranges from 3.4% to 9.4% for Kicer, and from 2.5% to 12.4% for SUVR. Moreover, we found good reproducibility of the data analysis by the automated pipeline (in the whole striatum for the Kicer: ICC for the controls = 0.71, ICC for the psychotic patients = 0.88). From the demographic and experimental variables assessed, gender was found to most influence striatal dopamine synthesis capacity (F = 10.7, p <0.001), with women showing greater dopamine synthesis capacity than men, while the effects of weight, age, injected radioactivity, and scanner, varied by brain region and parameter of interest. ConclusionsCombining information from different neuroimaging studies has allowed us to test comprehensively the automated pipeline for quantification of dopamine synthesis capacity using FDOPA PET data and to validate its replicability and reproducibility performances on a large sample size. This validation process is a necessary methodological step for the development of the clinical application of FDOPA PET as precision medicine biomarker. The proposed infrastructure is generalisable behind the FDOPA radiotracer.

bioengineering↗

bioRxiv2022

Effects of low intensity pulsed ultrasound stimulation on metabolic lipolysis of adipocytes

Obesity is closely related to several metabolic diseases along with abnormal increase in fat cells. Reducing size and number of fat cells, a procedure known as lipolysis, may be used to prevent obesity as a potential therapy, which also requires fundamental understanding of the mechanisms of lipolysis at molecular level upon different types of stimulations. Here, we used low intensity pulsed ultrasound (LIPUS) stimulation to investigate underlying mechanisms of the activation of lipolysis and autophagy related genes and signaling pathways of adipocytes differentiated from 3T3-L1 cells. LIPUS with the center frequency of 2 MHz was applied for 10 minutes per day for three days. After LIPUS stimulation, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot were used to determine the regulation of lipolytic factors such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL). At RNA level, all three factors were upregulated while only MGL was upregulated at protein level, which presents slightly different activation pattern of lipolysis compared to widely used chemical stimulation. These results demonstrate that LIPUS stimulation can promote the lipolytic capacity of adipocytes in the differentiated state. The differences between transcriptional genes and metabolites were analyzed by transcript analysis and metabolomic profiling experiments. Cellular RNA-sequencing (RNA-Seq) showed an increase in lipolysis and immune-related genes and autophagy related genes after LIPUS stimulation. This study may provide an important experimental basis for the clinical applications and a fundamental understanding of the mechanisms of lipolysis using LIPUS stimulation.

bioengineering↗

bioRxiv2022

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase-dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance (EPR) spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that for DPPC membrane whose gel-to-fluid lipid phase transition occurs at 314 K, TEMPO is stabilized above (below) the bilayer interface if the membrane is in the gel (fluid) phase. For bilayers made of unsaturated lipids, DOPC and POPC, which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.

biophysics↗

bioRxiv2022

Allele-specific activation, enzyme kinetics, and inhibitor sensitivities of EGFR exon 19 deletion mutations in lung cancer

Oncogenic mutations within the epidermal growth factor receptor (EGFR) are found in 15-30% of all non-small cell lung carcinomas. The term exon 19 deletion (ex19del) is collectively used to refer to more than 20 distinct genomic alterations within exon 19 that comprise the most common EGFR mutation subtype in lung cancer. Despite this heterogeneity, clinical treatment decisions are made irrespective of which EGFR ex19del variant is present within the tumor, and there is a paucity of information regarding how individual ex19del variants influence protein structure and function. Herein, we identify allele-specific functional differences among ex19del variants attributable to recurring sequence and structure motifs. We build all-atom structural models of 60 ex19del variants identified in patients and combine ~400 s of molecular dynamics simulations with biochemical and biophysical experiments to analyze three ex19del mutations (E746_A750, E746_S752>V, and L747_A750>P). We demonstrate that sequence variation in ex19del alters oncogenic cell growth, dimerization propensity, and tyrosine kinase inhibitor (TKI) sensitivity. We show that in contrast to E746_A750 and E746_S752>V, the L747_A750>P variant forms highly active ligand-independent dimers. E746_S752>V displays the least TKI sensitivity among the variants tested, which enzyme kinetic analysis and TKI inhibition experiments suggest is due to increased ATP Km relative to the common E746_A750 variant. Through these analyses, we propose an expanded framework for interpreting ex19del variants and new considerations for therapeutic intervention. SignificanceEGFR mutations are detected in approximately 30% of all lung adenocarcinomas, and the most common EGFR mutation occurring in ~50% of patients is termed "exon 19 deletion" (ex19del). Despite the existence of dozens of different genomic variants comprising what is generically referred to clinically as ex19del, clinicians currently do not distinguish between ex19del variants in considering treatment options, and the differences between ex19del variants are largely unstudied in the broader scientific community. Herein, we describe functional differences between distinct EGFR ex19del variants attributable to the structural features of each variant. These findings suggest a possible explanation for observed differences in patient outcomes stratified by ex19del subtype and reinforce the need for allele-specific considerations in clinical treatment decision making.

cancer biology↗

bioRxiv2022

IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) limits autoimmune inflammation by regulating myeloid cell activation and metabolic activity

Besides anti-viral functions, Type I IFN expresses potent anti-inflammatory properties and is being widely used to treat certain autoimmune conditions, such as multiple sclerosis (MS). In murine model of MS, experimental autoimmune encephalomyelitis (EAE), administration of IFN{beta} effectively attenuates the disease development. However, the precise mechanisms underlying the treatment remain elusive. In this study, we report that IFN-induced protein with tetratricopeptide repeats 2 (Ifit2), a type I and type III IFN-stimulated gene, plays a previously unrecognized immune regulatory role during autoimmune neuroinflammation. Mice deficient in Ifit2 display greater susceptibility to EAE and escalated immune cell infiltration in the central nervous system. Ifit2 deficiency is also associated with microglial activation and increased myeloid cell infiltration. Unexpectedly, myelin debris clearance and the subsequent remyelination is impaired in Ifit2-/- CNS tissues. Clearing myelin debris is an important property of reparative M2 type myeloid cells to promote remyelination. Indeed, we observed that bone marrow derived macrophages, CNS infiltrating myeloid cells, and microglia from Ifit2-/- mice express cytokine and metabolic genes associated with proinflammatory M1 type subsets. Taken together, our findings uncover a novel regulatory function of Ifit2 in autoimmune inflammation in part by modulating myeloid cell function and metabolic activity.

immunology↗

bioRxiv2022

Molecular profiling of XPO1 inhibitor and gemcitabine-nab-paclitaxel combination in cellular and LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) pancreatic cancer model

The majority of pancreatic ductal adenocarcinoma (PDAC) patients experience disease progression while on treatment with gemcitabine and nab-Paclitaxel (GemPac) treatment indicating the need for more effective combinations for this recalcitrant disease. Earlier we showed that nuclear exporter protein exportin 1 (XPO1) is a valid therapeutic target in PDAC and the selective inhibitor of nuclear export (SINE) selinexor (Sel), synergistically enhances the efficacy of GemPac in pancreatic cancer cells, spheroids, patient derived tumors and had promising activity in a phase I study in patients with PDAC. Here we investigated the mechanisms of synergy by molecular profiling of Sel or Sel-GemPac treated PDAC cells, in vitro and by utilizing genetically modified LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) mouse model. In KPC model, Sel given with GemPac at a sub-MTD dose enhanced the survival compared to controls (p < 0.05). Molecular analysis of residual KPC tumors showed re-organization of tumor stromal architecture, suppression of proliferation and nuclear retention of tumor suppressors. Single cell nuclear RNA sequencing (snRNAseq) revealed significant loss of cellular clusters in the Sel-GemPac treated mice including CD44 stem cell population. RNA-seq, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) analysis showed inhibition of several tumor promoting molecules. Prioritized RNA-seq identified molecules were validated in in vitro or in the PDAC patient samples through siRNA mediated silencing, quantitative gene expression, cytotoxicity assays and confirmed their role in observed synergy. Sel or Sel-GemPac caused broad penetration in PDAC supporting signaling networks.

cancer biology↗

bioRxiv2022

A versatile pattern-forming cortical circuit based on Rho, F-actin, Ect2 and RGA-3/4

Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner and, when co-expressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces a range of cortical behaviors from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho and F-actin form the core of a circuit that drives a diverse range of cortical behaviors, and demonstrate that the immature oocyte is a powerful model for characterizing these dynamics. SummaryMichaud et al. identify Ect2 and RGA-3/4 as core components of the cortical excitability circuit associated with cytokinesis. Additionally, they demonstrate that the immature Xenopus oocyte is a powerful model for characterizing excitable dynamics.

cell biology↗

bioRxiv2022

A method to estimate the effective point spread function of static single molecule localization microscopy images

Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent effective point spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from factors such as drift and drift correction algorithms. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labelled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. STATEMENT OF SIGNIFICANCESingle molecule localization microscopy (SMLM) is a class of imaging methods that resolve fluorescently labeled structures beyond the optical resolution limit of visible light. SMLM detects stochastically blinking labels over time, and localizes each blink with precision of order 10 nm. The effective resolution depends on factors such as signal-to-noise ratio, localization algorithm, and several post-processing steps such as stage drift correction. We present a method to evaluate this effective resolution by taking advantage of temporal correlations of fluorophore blinking to separate the distribution of pairs of localizations from the same molecule from those from different molecules. The method is robust on useful timescales for a range of SMLM probes.

biophysics↗

bioRxiv2022

Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize and Purify a Detergent-Sensitive Membrane Protein Assembly

The peptidisc membrane mimetic enables global reconstitution of the bacterial membrane proteome into water-soluble detergent-free particles, termed peptidisc libraries. We present here a method that combines peptidisc libraries and chromosomal-level gene tagging technology with affinity purification and mass spectrometry (AP/MS) to stabilize and identify fragile membrane protein complexes that exist at native expression levels. This method circumvents common artifacts caused by bait protein overproduction and protein complex dissociation due to lengthy exposure to detergents during protein isolation. Using the E. coli Sec system as a case study, we identify an expanded version of the translocon, termed the HMD complex, consisting of 9 different integral membrane subunits. This complex is stable in peptidiscs but dissociates in detergent. Guided by this native-level proteomic information, we design and validate a procedure that enables purification of the HMD complex with minimal protein dissociation. These results highlight the utility of peptidiscs and AP/MS to discover and stabilize fragile membrane protein assemblies.

biochemistry↗

bioRxiv2022

AIMP1-derived peptide secreted from hair follicle stem cells activates dermal papilla cells to promote hair growth

Hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs) are crucial in the biogenesis and maintenance of hair follicles (HFs). In this study, a fragment derived from aminoacyl-tRNA synthetase-interacting multifunctional protein1 (AIMP1) was secreted from HFSCs to activate DPCs to maintain hair follicle homeostasis. A histological analysis revealed that AIMP1 levels in hair follicles decreased with hair loss. Hair regrowth in AIMP1-induced mice was faster than that in non-induced mice. Deletion mapping revealed 41 amino acids (TN41, aa 6-46) as the active region of AIMP1. The N-terminal peptide fragment of AIMP1 generated by MMP1 was secreted from Wnt-treated HFSCs to activate DPCs via FGFR2. TN41 activated Akt and ERK, increased {beta}-catenin, and enhanced DPCs activation. TN41 also promoted hair shaft elongation in cultured human hair follicles and improved the hair-inducing activity of cultured DPC spheroids. In summation, the AIMP1 fragment secreted from HFSCs appears to stimulate active hair regrowth through activating DPCs.

cell biology↗

bioRxiv2022

Reactive astrocytes transduce blood-brain barrier dysfunction through a TNFα-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function in brain microvascular endothelial cells (BMECs). Transformation of astrocytes to a reactive state in response to injury and disease can be protective or harmful to BBB function, but the underlying mechanisms for these effects remain mostly unclear. Using a human induced pluripotent stem cell (iPSC)-derived coculture model of BMEC-like cells and astrocytes, we found that tumor necrosis factor alpha (TNF) transitions astrocytes to an inflammatory reactive state through activated STAT3 signaling, whereby the resultant astrocytes disrupt passive BBB function and induce vascular cell adhesion molecule 1 (VCAM-1) expression in the BMEC-like cells. These associations between inflammatory reactive astrocytes, STAT3 activation, and vascular VCAM-1 expression were corroborated in human postmortem tissue. Bioinformatic analyses coupled with CRISPR interference techniques in the iPSC model revealed that inflammatory reactive astrocytes transduce BBB disruption in part through SERPINA3, which encodes alpha 1-antichymotrypsin (1ACT), a secreted serine protease inhibitor associated with aging, neuroinflammation, and Alzheimers disease. In murine ex vivo cortical explant cultures, shRNA-mediated silencing of Serpina3n in astrocytes reduced vascular VCAM-1 expression after TNF challenge. Further, direct treatment with recombinant Serpina3n in both ex vivo explant cultures and the brain in vivo (via intracerebroventricular injection into wild-type mice) was sufficient to induce vascular VCAM-1 expression and reduce tight junction integrity. Overall, our results define the TNF-STAT3 signaling axis as a driver of an inflammatory reactive astrocyte subtype responsible for BBB dysfunction. Our results also identify 1ACT as an explicit mediator of BBB damage and suggest that inhibition of 1ACT expression or activity could represent a therapeutic avenue for reversing BBB deficits in aging and neurodegenerative disease.

neuroscience↗

bioRxiv2022

Exponential magnetophoretic gradient for the direct isolation of basophils from whole blood in a microfluidic system

Despite their rarity in peripheral blood, basophils play important roles in allergic disorders and other diseases including sepsis and COVID-19. Existing basophil isolation methods require many manual steps and suffer from significant variability in purity and recovery. We report an integrated basophil isolation device (i-BID) in microfluidics for negative immunomagnetic selection of basophils directly from 100 L of whole blood within 10 minutes. We use a simulation-driven pipeline to design a magnetic separation module to apply an exponentially increasing magnetic force to capture magnetically tagged non-basophils flowing through a microtubing sandwiched between magnetic flux concentrators sweeping across a Halbach array. The exponential profile captures non-basophils effectively while preventing their excessive initial buildup causing clogging. The i-BID isolates basophils with a mean purity of 93.9%{+/-}3.6% and recovery of 95.6%{+/-}3.4% without causing basophil degradation or unintentional activation. Our i-BID has the potential to enable basophil-based point-of-care diagnostics such as rapid allergy assessment.

immunology↗

bioRxiv2022

Foldseek: fast and accurate protein structure search

As structure prediction methods are generating millions of publicly available protein structures, searching these databases is becoming a bottleneck. Foldseek aligns the structure of a query protein against a database by describing the amino acid backbone of proteins as sequences over a structural alphabet. Foldseek decreases computation times by four to five orders of magnitude with 86%, 88% and 133% of the sensitivities of DALI, TM-align and CE, respectively.

bioinformatics↗

bioRxiv2022

AAA+ protease-adaptor structures reveal altered conformations and ring specialization

ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here, we present high-resolution cryo-EM structures of ClpAPS complexes showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two structural classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated and tucked away from the channel, and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies demonstrate new structures and functions for key AAA+ elements. In addition to ClpAPS delivery, pore-loop tucking may be used broadly by AAA+ unfoldases, for example during enzyme pausing/unloading.

biophysics↗

bioRxiv2022

Establishment of physiologically relevant oxygen gradients in microfluidic organ chips

In vitro models of human organs must accurately reconstitute oxygen concentrations and gradients that are observed in vivo to mimic gene expression, metabolism, and host-microbiome interactions. Here we describe a simple strategy to achieve physiologically relevant oxygen tension in a two-channel human small intestine-on-a-chip (Intestine Chip) lined with primary human duodenal epithelium and intestinal microvascular endothelium in parallel channels separated by a porous membrane while both channels are perfused with oxygenated medium. This strategy was developed using computer simulations that predicted lowering the oxygen permeability of poly-dimethlysiloxane (PDMS) chips in specified locations using a gas impermeable film will allow the cells to naturally decrease the oxygen concentration through aerobic respiration and reach steady-state oxygen levels < 36 mm Hg (< 5%) within the epithelial lumen. The approach was experimentally confirmed using chips with embedded oxygen sensors that maintained this stable oxygen gradient. Furthermore, Intestine Chips cultured with this approach supported formation of a villus epithelium interfaced with a continuous endothelium and maintained intestinal barrier integrity for 72 h. This strategy recapitulates in vivo functionality in an efficient, inexpensive, and scalable format that improves the robustness and translatability of Organ Chip technology for studies on microbiome as well as oxygen sensitivity.

bioengineering↗

bioRxiv2022

Inhibitor of the nuclear transport protein XPO1 enhances the anticancer efficacy of KRAS G12C inhibitors in preclinical models of KRAS G12C mutant cancers

The identification of molecules that can bind covalently to KRAS G12C and lock it in an inactive GDP-bound conformation has opened the door to targeting KRAS G12C selectively. These agents have shown promise in preclinical tumor models and clinical trials. FDA has recently granted approval to sotorasib for KRAS G12C mutated non-small cell lung cancer (NSCLC). However, patients receiving these agents as monotherapy may not respond and generally develop drug resistance over time. This necessitates the development of multi-targeted approaches that can potentially sensitize tumors to KRAS inhibitors. We generated KRAS G12C inhibitor-resistant cell lines and observed that they exhibit sensitivity toward selinexor, a selective inhibitor of nuclear export protein exportin1 (XPO1), as a single agent. KRAS G12C inhibitor MRTX1257 in combination with selinexor suppressed the proliferation of KRAS G12C mutant cancer cell lines MiaPaCa-2 and NCI-H2122 in a synergistic manner. Moreover, combined treatment of selinexor with KRAS G12C inhibitors resulted in enhanced spheroid disintegration, reduction in the number and size of colonies formed by G12C mutant cancer cells. A combination of selinexor with KRAS G12C inhibitors potentiated the inhibition of KRAS expression in MiaPaCa-2 cells. NF-kB protein expression was also markedly reduced by selinexor and MRTX1257 combination. In an in vivo KRAS G12C cell-derived xenograft model, oral administration of a combination of selinexor and sotorasib was demonstrated to reduce tumor burden and enhance survival. In conclusion, we have shown that the nuclear transport protein XPO1 inhibitor can enhance the anticancer activity of KRAS G12C inhibitors in preclinical cancer models. SignificanceIn this study, combining nuclear transport inhibitor selinexor with KRAS G12C inhibitors has resulted in potent antitumor effects in preclinical cancer models. This can be an effective combination therapy for cancer patients that do not respond or develop resistance to KRAS G12C inhibitor treatment.

cancer biology↗