STING (stimulator of interferon genes) agonists are promising innate immune therapies and can be synergized with adaptive immune checkpoint blockade therapies for cancer treatment, but their effectiveness is limited by t...STING (stimulator of interferon genes) agonists are promising innate immune therapies and can be synergized with adaptive immune checkpoint blockade therapies for cancer treatment, but their effectiveness is limited by the toxicity to activated T cells. How STING agonists such as cGAMP and its analogs enter and induce STING activation and toxicity in T cells is unclear despite known transporters for other cell types. Here, we identify the cationic amino acid transporter SLC7A1 as a cGAMP transporter in activated primary mouse and human T cells. T cells upregulate this transporter upon activation to meet their high metabolic demand, but this comes at the cost of enabling increased transport and toxicity of cGAMP. We identified distinct residues in SLC7A1 that mediate cGAMP and arginine activity, suggesting that cGAMP transport may be separable from arginine uptake. These findings suggest that modulation of SLC7A1 may influence T cell susceptibility to cGAMP and its analogs.
Insulin secretion can be stimulated by immune and neuronal processes prior to a rise in blood glucose, exemplified by the cephalic phase of insulin response in the anticipation of food. Pancreatic α-cells prevent hypogly...Insulin secretion can be stimulated by immune and neuronal processes prior to a rise in blood glucose, exemplified by the cephalic phase of insulin response in the anticipation of food. Pancreatic α-cells prevent hypoglycemia by releasing glucagon. Here, we identified α-cells as critical mediators of IL-1β- and cholinergic agonist-driven insulin secretion. Cholinergic blockade prevented glucagon-stimulated insulin secretion in mice. Selective ablation of α-cells abolished cephalic phase insulin release. Islets from α-cell-deficient mice also failed to secrete insulin in response to IL-1β or muscarinic receptor activation. However, glucagon, acting on glucagon and GLP-1 receptors, rescued this insulin-stimulatory response. Mechanistically, intracellular Ca mobilization at fasting glucose mediated cholinergic and IL-1β-stimulated insulin release. Short-term high-fat diet impaired glucagon-induced insulin secretion in vivo, while isolated islets showed increased insulin secretion after cholinergic stimulation versus chow-fed controls. These findings reveal α-cell-derived glucagon as a gatekeeper of immune and neuronal control of insulin secretion at fasting glucose.
Abnormal DNA methylation occurs in Huntington's disease (HD, but the underlying mechanisms remain unclear. Using a knock-in pig model, we identify significant alterations in 5mC and 5hmC levels linked to neural genes. TE...Abnormal DNA methylation occurs in Huntington's disease (HD, but the underlying mechanisms remain unclear. Using a knock-in pig model, we identify significant alterations in 5mC and 5hmC levels linked to neural genes. TET1, which converts 5mC to 5hmC, decreases prior to symptom onset in pigs-a change not observed in HD mice. TBP binding sites are abundant in the porcine and human TET1 promoter, but scarce in mice. Mutant huntingtin (mHTT) binds more strongly to TBP in HD pig brains, blocking TBP's access to the TET1 promoter, thereby reducing TET1 transcription and altering 5mC/5hmC patterns. Our findings reveal TET1 as a target of abnormal DNA methylation that contributes to selective neuronal vulnerability in HD pigs, highlighting the value of large mammalian models for studying disease pathogenesis.
Gillman AN, Birler CM, Vujjini RR
… +10 more, Fili M, McCarthy-Potter SA, Chen W, Broghammer MM, Hu G, Gartland MJ, Prakash M, Helverson A, Brown GG, Haim H
HIV-1 resistance to therapeutics can emerge through diverse mutational routes, yet the determinants guiding pathway selection in vivo remain unclear. Through comprehensive screening, we identify 18 mutations in the HIV-1...HIV-1 resistance to therapeutics can emerge through diverse mutational routes, yet the determinants guiding pathway selection in vivo remain unclear. Through comprehensive screening, we identify 18 mutations in the HIV-1 Env protein that enhance resistance to the FDA-approved small-molecule therapeutic temsavir. We then examine their occurrence in HIV-infected individuals who developed resistance on therapy. Interestingly, only a subset of the resistance-enhancing mutations emerged in vivo. On-treatment mutation frequencies correlate with their emergence rates in temsavir-untreated individuals and are governed by two parameters: (1) probability of mutation appearance, determined by the number and type of nucleotide changes required, and (2) probability of mutation persistence, determined by Env functional and immune fitness. Notably, non-neutralizing antibodies commonly elicited in HIV-infected individuals restrict emergence of multiple resistant forms, driving convergence to a narrow set of escape routes. These findings establish a quantitative framework for predicting therapeutic resistance and reveal how host immunity constrains viral evolution during treatment.
Fragile X syndrome (FX) is associated with sensory processing and learning deficits. Visual familiarity evokes persistent theta oscillations during passive stimulus presentation in the primary visual cortex (V1) and the...Fragile X syndrome (FX) is associated with sensory processing and learning deficits. Visual familiarity evokes persistent theta oscillations during passive stimulus presentation in the primary visual cortex (V1) and the hippocampus (HPC), which are impaired in V1 of FX. How does this activity change during active behavior? To address this, we performed Neuropixels recordings in V1, HPC, and the prefrontal cortex (PFC) during Go/No-Go visual discrimination behavior. Theta oscillations are reduced in FX in both V1 and HPC and are abolished during No-Go trials, correlating with excessive, incorrect licking behavior. In wild-type (WT) mice, V1 theta power strongly correlates with correct behavioral outcomes. PFC shows significantly reduced cue-related responses in FX. Together, these findings show loss of behavioral inhibition in FX correlated with attenuated theta activity in V1 and HPC and deficient top-down control from PFC. This work sheds light on circuit-level impairments underlying behavioral deficits in FX for potential therapeutic interventions.
Zhang Y, Zhou X, Wei Y
… +22 more, Zhang H, Zhao Y, Tong H, Jiang Z, Li G, Jiang Y, Yin X, Li S, Lin N, Feng L, Zhou H, Kang Q, Qiu J, Wei J, Tan K, Yang X, Li R, Yang J, Tang S, Zhang S, Liu J, Ma X
Immune checkpoint blockade (ICB) shows limited efficacy in pancreatic ductal adenocarcinoma (PDAC). Here, we demonstrate that ICB-induced IFN-γ signaling upregulates H2-T23 on pancreatic tumor cells, which interacts with...Immune checkpoint blockade (ICB) shows limited efficacy in pancreatic ductal adenocarcinoma (PDAC). Here, we demonstrate that ICB-induced IFN-γ signaling upregulates H2-T23 on pancreatic tumor cells, which interacts with NKG2A on NK cells to induce NK cell senescence through activation of p38 MAPK and STAT1/3 pathways. This impairs NK cell cytotoxicity and restricts antitumor immunity. Dual blockade of PD-1 and NKG2A effectively prevents NK cell senescence, restores NK cell function, and enhances antitumor immunity. Mechanistically, the combination therapy promotes NK cell-derived CCL5 and facilitates CD8 T cell recruitment in an NK cell-dependent manner, thereby activating both innate and adaptive immunity. Analysis of single-cell sequencing data across nine cancer types further revealed increased NK cell senescence after immunotherapy, suggesting a potentially common pan-cancer mechanism. These findings identify NK cell senescence as a key mechanism underlying immunotherapy resistance and support dual targeting of PD-1 and NKG2A as a promising therapeutic strategy for PDAC.
Interferon-stimulated gene 15 (ISG15), composed of two ubiquitin-like domains, plays a critical role in antiviral immunity. Although the ubiquitination mechanism is well established, the mechanisms governing ISG15 transf...Interferon-stimulated gene 15 (ISG15), composed of two ubiquitin-like domains, plays a critical role in antiviral immunity. Although the ubiquitination mechanism is well established, the mechanisms governing ISG15 transfer, particularly from E2 to E3 and subsequent lysine conjugation, remain unknown. Here, we reveal that UbcH8(E2)∼ISG15 exhibits striking specificity for HECT-family E3 ligases (particularly HERC5) but is inactive with RING or RBR E3. In contrast, UbcH8∼Ub preferentially engages RBR E3, indicating a switched E2-E3 specificity depending on the conjugated ubiquitin-like modifier. Structural and biochemical studies uncover how a closed conformation of UbcH8∼ISG15 enables trans-thiolation mediated by selective HECT-family E3 ligases. We further demonstrate that HERC5's C-lobe specifically recognizes donor ISG15 for lysine conjugation, explaining its exclusive ISGylation activity and lack of ubiquitination function. These findings delineate the molecular basis of ISG15 conjugation and reveal how its pathway has evolved distinct mechanisms from ubiquitination, offering new avenues for therapeutic intervention.
Bacteriophages (phages) are abundant members of the gut microbiota and regulators of bacterial communities. During homeostasis, gut phage communities are longitudinally stable and lysogenic replication is dominant. In ch...Bacteriophages (phages) are abundant members of the gut microbiota and regulators of bacterial communities. During homeostasis, gut phage communities are longitudinally stable and lysogenic replication is dominant. In chronic gut inflammatory disorders, such as inflammatory bowel diseases (IBDs), there are alterations in phage diversity, which may result from changes in phage replication cycles. Here, we use a combination of in vitro, simplified community, and whole-community bioinformatics approaches to investigate whether prophage induction contributes to these alterations. We identify several compounds associated with gut inflammation that induce prophages from commensal gut bacterial isolates. Analyzing data from two mouse models of colitis, we observe that shifts in the composition of temperate phages occur over the course of inflammation, supporting a switch from lysogenic to lytic replication. Collectively, our observations support the idea that prophage induction contributes to alterations in the phageome associated with intestinal inflammation.
Jafree DJ, Russell LG, Stathopoulou A
… +25 more, O'Riordan C, Rowan CJ, White AT, Kolatsi-Joannou M, Price KL, Ivins S, Ridge LA, Roberts C, Rose A, Webb S, Vijayabaskar MS, Chandler JC, Wilson L, Pomeranz G, Moulding D, Davis B, Mitchell H, Siegenthaler J, Haniffa M, Woolf AS, Riley PR, Ruhrberg C, Scambler PJ, Rosenblum ND, Long DA
Lymphatic vessels perform diverse functions, ranging from fluid homeostasis to immune regulation, and arise from multiple cellular origins to form organ-specific networks. Despite their importance in kidney disease and t...Lymphatic vessels perform diverse functions, ranging from fluid homeostasis to immune regulation, and arise from multiple cellular origins to form organ-specific networks. Despite their importance in kidney disease and transplant immunity, the origins of kidney lymphatics are unknown. Using genetic lineage tracing in mice, we identify two origins of kidney lymphatics. Most kidney lymphatics arise from a Tie2 endothelial origin shared by other organs. However, Osr1 mesoderm generates approximately 15% of kidney lymphatics, without contributing to heart, mesentery, and skin lymphatics. Interrogating single-cell transcriptomics data of mice and humans reveals lymphatic progenitors within Osr1 mesoderm. Lymphatic clusters forming by de novo assembly originate from both Osr1 and Tie2 lineages. Deleting the lymphatic specification gene Prox1 in Osr1 mesoderm reduces lymphatic cluster number, impairing overall lymphatic network complexity, with lower glomerular number. Thus, an Osr1 mesodermal origin contributes to organ-specific lymphatic assembly, with consequences for kidney health, disease, and regeneration.
Suppressive myeloid cells play a central role in cancer escape from anti-tumor immunity and exert multiple pro-tumoral activities, including the promotion of cancer cell survival, invasion, and metastasis. Recent evidenc...Suppressive myeloid cells play a central role in cancer escape from anti-tumor immunity and exert multiple pro-tumoral activities, including the promotion of cancer cell survival, invasion, and metastasis. Recent evidence suggests that certain subsets may also influence tumor heterogeneity and cellular plasticity. However, their role in cancer stem cell promotion and plasticity and the underlying molecular mechanisms have yet to be deciphered. Here we demonstrate that human immunosuppressive myeloid cells, generated in vitro or isolated from patients with breast cancer, induce cancer stem-like cells exhibiting mesenchymal features. This cancer-stemness-inducing function was restricted to a CD52-expressing myeloid subset. Single cell transcriptomic- and surface proteome-based interactome analyses identify membrane-bound TGF-β1 as a potential mediator of this effect. Functional inhibition of the TGF-β1 pathway blocks stem-like feature induction by suppressive myeloid cells. These results provide insights into the cancer stemness-promoting capacity of suppressive myeloid cells and its associated molecular mechanisms in breast cancer.
Autophagy is a catabolic process that degrades cytoplasmic materials and is controlled by nutrient availability and signaling. The plasma membrane-associated pyruvate-solute carrier hermes (hrm) is required for regulatio...Autophagy is a catabolic process that degrades cytoplasmic materials and is controlled by nutrient availability and signaling. The plasma membrane-associated pyruvate-solute carrier hermes (hrm) is required for regulation of the mechanistic target of rapamycin (mTOR) signaling and the activation of autophagy during development. Here, we screen for pyruvate-influencing genes that suppress the hrm mutant phenotype. We show that the inhibitory effect of hrm loss on autophagy depends on pyruvate transport into mitochondria and the Krebs cycle. Loss of hrm results in an increase in reactive oxygen species (ROS), and attenuation of the increase in ROS is sufficient to suppress the effects of hrm loss on autophagy and mTOR signaling. Importantly, we show that in adult animals, loss of hrm results in decreased lifespan, with defects in autophagy in intestine tissues. These results link a plasma membrane pyruvate carrier to mitochondrial pyruvate metabolism, ROS, autophagy, and organismal health.
Zhou L, Yu Z, Lin S
… +25 more, Jiang Y, Gao J, Ma Y, Jiang W, Liang S, Chen Y, Zhang Y, Lin Y, Liang M, Dai J, Zhang L, Xiao Y, Li T, Kong Z, Liu Q, He S, Wu Y, Yuan Q, Liu F, Zhang J, Zheng Q, Yu H, Gu Y, Li S, Xia N
Broadly neutralizing antibodies (bnAbs) are essential for the development of vaccines and therapeutics against rapidly evolving pathogens like HIV and SARS-CoV-2, yet traditional discovery methods remain technically chal...Broadly neutralizing antibodies (bnAbs) are essential for the development of vaccines and therapeutics against rapidly evolving pathogens like HIV and SARS-CoV-2, yet traditional discovery methods remain technically challenging and time consuming. Here, we introduce ClonoDeep, an AI-powered platform that integrates public antibody clonotypes with a sequence-based deep learning model to directly identify bnAbs from a large-scale immune repertoire, independent of antigen-specific immunization. Applied to SARS-CoV-2 repertoires, ClonoDeep identified 18 clonotype-derived antibody candidates; 83% of the candidates were neutralizing antibodies, and 8 of these antibodies demonstrated broad neutralization across variants. Structural analysis revealed that somatic hypermutations at HCDR3 His107/Gly109 are key enhancers of the binding affinity and neutralizing breadth. Extending to HIV, ClonoDeep uncovered three previously unreported bnAbs from non-HIV cohorts, indicating that rare bnAb-like precursors exist in non-HIV cohort repertoires. ClonoDeep establishes a high-throughput computational approach for mining neutralizing antibodies from antibody repertoires shaped by non-pathogen-specific immunity and provides design principles to guide vaccine strategies against genetically diverse pathogens.
IL-10-signaling-deficiency-induced macrophage hyperactivation drives epithelial barrier disruption and severe colitis, but its role in colitis-associated colorectal cancer (CAC) remains unclear. Here, we report that macr...IL-10-signaling-deficiency-induced macrophage hyperactivation drives epithelial barrier disruption and severe colitis, but its role in colitis-associated colorectal cancer (CAC) remains unclear. Here, we report that macrophage-specific IL-10Rα deletion aggravates AOM-DSS-induced CAC in mice. These IL-10Rα-deficient macrophages exhibited a pro-inflammatory phenotype and secreted excessive IL-6 to stimulate intestinal epithelial cell (IEC) proliferation and tumorigenesis via the IL-6/p-STAT3 pathway. Genetic ablation or neutralization of macrophage-specific IL-6Rα reduced IEC p-STAT3 levels and tumor burden, whereas epithelial IL-6Rα deficiency or sgp130 treatment did not. IEC-BMDM co-culture and imaging flow cytometry identify a previously unrecognized macrophage-derived IL-6 trans-presentation as the dominant driver of IEC proliferation and tumorigenesis, rather than classical/trans-signaling. Targeting macrophage IL-6Rα in combination with PD-L1 blockage exerts complementary effects in CAC. Collectively, our data reveal that macrophage-specific IL-10 signaling protects against CAC by suppressing macrophage-dependent IL-6 trans-presentation, which drives excessive IEC proliferation and tumor development.
Lin SZ, Chen Y, Wu C
… +62 more, Sun WH, Li Z, Chen HC, Wang JY, Ji CM, Li SB, Wang ZW, Tsai WC, Ma XQ, Lan SR, Zhang FP, Xie YC, Yao L, Zhang Y, Lü MM, Zhang JJ, Zhang DY, Ye YQ, Yu X, Xu SS, Ma ZH, Ding GC, Cao GQ, He ZM, Wu PF, Lin KM, Liu AQ, Lin YQ, Ruan SN, Liu B, Cao SJ, Zhou LL, Li M, Shuai P, Hou XL, Wu YH, Li N, Xiong S, Hao Y, Zhou Z, Liu XD, Zuo DD, Li J, Wang P, Zhang J, Liu DK, Chen GZ, Huang J, Huang MZ, Li YY, Zheng QY, Zhao XW, Zhao X, Zhong WY, Zhang XW, Xia ZB, Yu Y, Liu ZW, Zheng HK, Ming R, Van de Peer Y, Liu ZJ
Cupressaceae , a gymnosperm family, draws attention due to its controversial phylogenetic position. Here, we present a comprehensive genome analysis of Chinese fir (Cunninghamia lanceolata), a Cupressaceae species, to en...Cupressaceae , a gymnosperm family, draws attention due to its controversial phylogenetic position. Here, we present a comprehensive genome analysis of Chinese fir (Cunninghamia lanceolata), a Cupressaceae species, to enhance our understanding of gymnosperm evolution. The 11.24 Gb assembled genome, shaped by inefficient long terminal repeat removal, offers insights into its phylogenetic position. Phylogenetic analysis refines gymnosperm relationships between Cycads-Ginkgo and their relation to Gnetales-Pinaceae. Whole-genome duplication (WGD) analysis reveals no evidence for an ancient polyploidization event in the lineage of C. lanceolata, and confirms a seed-plant-shared WGD event. We also explore genomic evidence to explain the population history and adaptability of C. lanceolata, including potential glacial refugia, dispersal centers, and unique sterility. Furthermore, the refined (A)B(C) model for reproductive organ development in C. lanceolata has broader applications across gymnosperms. This study provides a valuable genome sequence and contributes to the understanding of gymnosperm evolution.
Complex molecular mechanisms underlie the heterogeneity of microglia immune functions in neurodegenerative diseases. Here, we report on the transcriptional mechanisms that control microglial activities associated with de...Complex molecular mechanisms underlie the heterogeneity of microglia immune functions in neurodegenerative diseases. Here, we report on the transcriptional mechanisms that control microglial activities associated with demyelination in mice. Using flow cytometry, microscopy, and RNA-seq, we identify two dominant states of inflammatory microglia: Clec7aCD229CD11c microglia, which are prone to proliferation and express high mRNA levels of Fn1 and Vegfa, and Clec7aCD229CD11c microglia, which are characterized by prominent mRNA expression of tissue-remodeling and antigen presentation effectors. Achieving these states implicates genome-wide nucleosome remodeling, which is driven by state-dependent stimulation of transcription factors such as Pu.1, AP-1, C/ebp, Mef2, and Egr2. Notably, an H3K27me3-based gatekeeping mechanism controls expression of key regulators, including Egr2. Mechanistically, we validate the relevance of Trem2, Mef2a, and Egr2 to the inflammatory microglial polarization process. Therefore, distinct configurations of signals, along with chromatin remodeling, orchestrate transcription in microglia to support their immune activities in the context of demyelination.
Microglia are central nervous system (CNS)-resident macrophages, with key roles in immune surveillance, phagocytosis, and synaptic pruning. Yolk sac-derived microglia show minimal turnover from hematopoietic stem/progeni...Microglia are central nervous system (CNS)-resident macrophages, with key roles in immune surveillance, phagocytosis, and synaptic pruning. Yolk sac-derived microglia show minimal turnover from hematopoietic stem/progenitor cells (HSPCs) under steady-state conditions in mice. However, clinical benefits observed in patients receiving HSPC gene therapies for CNS disorders suggest functional integration of HSPC-derived cells. To investigate microglia replacement and the impact of clonal hematopoiesis (CH) on microglia, we analyzed microglia in rhesus macaques receiving barcoded or CRISPR-edited (TET2-mutant) HSPC transplants. We found that <2% microglia were derived from HSPCs many years following transplant, with no evidence of enhanced replacement in CH. The rare HSPC-derived tissue-resident cells exhibited a macrophage-like gene expression profile. Our results demonstrate limited long-term microglia replacement from adult HSPCs, even with CH, contrasting prior human studies. This work provides insights into microglia ontogeny and informs strategies for CNS-targeted HSPC gene therapies and interpretation of CH-related neuroprotection.
Immunometabolic remodeling drives adaptation to long-term high-altitude exposure (LTHAE), yet the underlying mechanisms remain elusive. By integrating single-cell transcriptomics and metabolomics from 46 lowlanders follo...Immunometabolic remodeling drives adaptation to long-term high-altitude exposure (LTHAE), yet the underlying mechanisms remain elusive. By integrating single-cell transcriptomics and metabolomics from 46 lowlanders following a 90-day LTHAE, we identified a strategy of "innate activation and adaptive suppression." Neutrophils exhibited enhanced maturation and phagocytosis, whereas adaptive immunity was dampened, characterized by suppressed B cell function and T cell responsiveness. Metabolically, LTHAE induced systemic shifts in steroid and amino acid metabolism associated with immune remodeling. Furthermore, multi-omics integration indicated a conserved upregulation of the glycolysis-TCA-OXPHOS axis across immune lineages, a metabolic adaptation supported by enzymatic assays in hematopoietic tissues of a hypobaric hypoxia mouse model. Collectively, these findings provide integrated insights into immune-metabolic landscape remodeling and suggest a potential mutual regulatory relationship between immune and metabolic state following LTHAE, offering a molecular foundation for high-altitude adaptation research.
Bonnard B, Chatefau A, Dourthe C
… +12 more, Di Tommaso S, Dupuy JW, Mahouche I, Solorzano J, Février L, Le Bras M, Raymond AA, Moreau V, Jabouille A, Blangy A, Martineau Y, Saltel F
Locomotor skills arise early in life and are maintained throughout an animal's lifespan, yet how this continuity is achieved despite major neural remodeling remains unclear. Using Drosophila, which undergoes complete met...Locomotor skills arise early in life and are maintained throughout an animal's lifespan, yet how this continuity is achieved despite major neural remodeling remains unclear. Using Drosophila, which undergoes complete metamorphosis, we show that the activity of embryonically established dopaminergic neurons (DANs) is essential for locomotion across all developmental stages and adulthood. Through stage-specific behavioral assays, optogenetics, in vivo brain imaging, and fluorescent neuronal tracking, we identify a subset of ventral nervous system (VNS) DANs that modulate locomotor function throughout life. Transcriptomic analyses reveal that they maintain expression of developmental transcription factors. Knocking down these factors, particularly Antp and Pdm2, in post-mitotic VNS DANs reduces neurite arborization and impairs adult locomotion. These findings uncover a previously overlooked function for embryonic DANs and suggest that stable locomotion during nervous system maturation relies on persistent developmental regulator expression coupled with structural remodeling.
RAS inhibitors have demonstrated limited efficacy in KRAS-mutant colorectal cancer (CRC). Here, we evaluate RMC-7977, a RAS(ON) multi-selective inhibitor, which shows a heterogeneous response in a panel of preclinical CR...RAS inhibitors have demonstrated limited efficacy in KRAS-mutant colorectal cancer (CRC). Here, we evaluate RMC-7977, a RAS(ON) multi-selective inhibitor, which shows a heterogeneous response in a panel of preclinical CRC models. Inhibition of PI3K signaling and cyclin D1 (CCND1) expression are strongly correlated with response. We find that stratifying tumors based on the ERK dependence of cyclin D1, mediated by variability in CCND1 mRNA regulation, reveals two distinct resistance mechanisms. PI3K signaling plays a key role in driving persistent cyclin D1 expression in the less ERK-dependent tumors, and concurrent inhibition of RAS and PI3K can synergistically cause cell cycle arrest and promote apoptosis. However, in tumors with strongly ERK-dependent cyclin D1, vertical inhibition of RAS and MEK prevents feedback re-activation of the MAPK/ERK pathway and is more effective. In summary, we identify cyclin D1 as an important integrator and functional readout of upstream signaling that can inform optimal combination strategies for KRAS-mutant CRC.