Abstract

摘要

KRAS-specific inhibitors have shown promising antitumor effects, especially in non-small cell lung cancer, but limited efficacy in colorectal cancer (CRC) patients. Recent studies have shown that EGFR-mediated adaptive feedback mediates primary resistance to KRAS inhibitors, but the other resistance mechanisms have not been identified.

KRAS特异性抑制剂已显示出有希望的抗肿瘤作用，特别是在非小细胞肺癌中，但在结直肠癌（CRC）患者中疗效有限。最近的研究表明，EGFR介导的适应性反馈介导了对KRAS抑制剂的原发性耐药，但其他耐药机制尚未确定。

In this study, we investigated intrinsic resistance mechanisms to KRAS inhibitors using patient-derived CRC cells (CRC-PDCs). We found that KRAS-mutated CRC-PDCs can be divided into at least an EGFR pathway-activated group and a PI3K/AKT pathway-activated group. In the latter group, PDCs with PIK3CA major mutation showed high sensitivity to PI3K+mTOR co-inhibition, and a PDC with Her2 amplification with PIK3CA minor mutation showed PI3K-AKT pathway dependency but lost KRAS-MAPK dependency by cytoplasmic localization of KRAS.

在这项研究中，我们使用患者来源的CRC细胞（CRC-pDC）研究了对KRAS抑制剂的内在耐药机制。我们发现KRAS突变的CRC pDC可分为至少EGFR途径激活组和PI3K/AKT途径激活组。在后一组中，具有PIK3CA主要突变的PDC显示出对PI3K+mTOR共抑制的高度敏感性，并且具有PIK3CA次要突变的Her2扩增的PDC显示PI3K-AKT途径依赖性，但通过KRAS的细胞质定位丧失了KRAS-MAPK依赖性。

In the PDC, Her2 knockout restored KRAS plasma membrane localization and KRAS inhibitor sensitivity. The current study provides insight into the mechanisms of primary resistance to KRAS inhibitors, including aberrant KRAS localization..

在PDC中，Her2敲除恢复了KRAS质膜定位和KRAS抑制剂敏感性。目前的研究提供了对KRAS抑制剂原发性耐药机制的见解，包括异常的KRAS定位。。

Introduction

简介

The GTPase protein KRAS is commonly mutated in various cancer types, including pancreatic adenocarcinoma (PDAC), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC), occurring in 90%, 40%, and 20% of cases in each cancer type, respectively

GTPase蛋白KRAS通常在各种癌症类型中发生突变，包括胰腺癌（PDAC），结直肠癌（CRC）和非小细胞肺癌（NSCLC），分别发生在每种癌症类型的90%，40%和20%的病例中

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. Guanine nucleotide exchange factors and GTPase-activating proteins (GAPs) positively and negatively regulate the KRAS GTPase cycle from its inactive state (GDP-KRAS) to its active state (GTP-KRAS), respectively

鸟嘌呤核苷酸交换因子和GTPase激活蛋白（GAP）分别正向和负向调节KRAS GTPase循环从其非活性状态（GDP-KRAS）到活性状态（GTP-KRAS）

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. Codon 12–13 mutations usually impair the KRAS-GTP–GAP interaction, leading to constitutive activation of KRAS and its downstream signaling, including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways

密码子12-13突变通常会损害KRAS-GTP-GAP相互作用，导致KRAS及其下游信号的组成型激活，包括丝裂原活化蛋白激酶（MAPK）和磷脂酰肌醇-3激酶（PI3K）途径

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. While direct targeting of KRAS has long been challenging due to its high affinity for GTP binding and a lack of small pockets, recent efforts have succeeded in developing mutant KRAS-specific inhibitors

尽管由于KRAS对GTP结合的高亲和力和缺乏小口袋，直接靶向KRAS长期以来一直具有挑战性，但最近的努力已成功开发出突变的KRAS特异性抑制剂

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. For instance, KRAS-G12C, -G12D, and pan-KRAS inhibitors have been developed and evaluated in clinical settings

例如，KRAS-G12C，-G12D和泛KRAS抑制剂已经在临床环境中开发和评估

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. These KRAS inhibitors mainly bind to GDP-KRAS to block it from transitioning from inactive form to active GTP form, leading to the suppression of the KRAS effector pathway and elicitation of the antitumor effect. Although the first-leading KRAS-G12C inhibitors Sotorasib (AMG510) and Adagrasib (MRTX849) showed promising response rates (37% and 45%, respectively) in patients with KRAS-G12C-positive NSCLC, these inhibitors demonstrated unsatisfactory response rates (7% and 18%, respectively) in patients with KRAS-G12C-positive CRC, necessitating impediment to their US FDA-based approval for CRC treatment.

这些KRAS抑制剂主要与GDP-KRAS结合，阻止其从无活性形式转变为活性GTP形式，从而抑制KRAS效应通路并引发抗肿瘤作用。尽管第一个主要的KRAS-G12C抑制剂索托拉西布（AMG510）和阿达拉西布（MRTX849）在KRAS-G12C阳性NSCLC患者中显示出有希望的缓解率（分别为37%和45%），但这些抑制剂在KRAS-G12C阳性CRC患者中表现出不令人满意的缓解率（分别为7%和18%），因此需要阻碍其基于美国FDA的CRC治疗批准。

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. This highlights the need for research into the intrinsic resistance mechanisms to KRAS inhibitors for improved clinical efficiency in patients with CRC.

这突出表明需要研究KRAS抑制剂的内在耐药机制，以提高CRC患者的临床效率。

Intrinsic, adaptive, and acquired resistance to molecular targeting therapies emerges due to genetic and/or nongenetic alterations. Activating receptor tyrosine kinases (RTKs) or the PI3K pathway, reactivating MAPK signaling, or inducing epithelial-to-mesenchymal transition mediated intrinsic or adaptive resistance to KRAS inhibitors, which were able to enhanced therapeutic efficacy through the co-targeting of the essential molecules in these pathways in NSCLC or PDAC models.

由于遗传和/或非遗传改变，出现了对分子靶向疗法的内在，适应性和获得性抗性。激活受体酪氨酸激酶（RTKs）或PI3K途径，重新激活MAPK信号传导，或诱导上皮-间质转化介导的对KRAS抑制剂的内在或适应性抗性，这些抑制剂能够通过共同靶向这些途径中的必需分子来增强治疗功效。NSCLC或PDAC模型。

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. Activating epithelial growth factor receptor (EGFR) conferred intrinsic resistance to KRAS-G12C inhibitor through MAPK pathway reactivation in CRC models; combining anti-EGFR antibody cetuximab with KRAS inhibitor (e.g., sotolasib) showed synergistic effect in a preclinical study

激活上皮生长因子受体（EGFR）通过CRC模型中的MAPK途径再激活赋予对KRAS-G12C抑制剂的内在抗性；在临床前研究中，将抗EGFR抗体西妥昔单抗与KRAS抑制剂（例如索托拉西布）联合使用显示出协同作用

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. Notably, a clinical trial revealed that adagrasib and cetuximab combination therapy resulted in a markedly improved response rate (from 19% to 46%) in patients with CRC harboring KRAS-G12C, suggesting that this combination therapy could be a powerful therapeutic tool for this patient population

值得注意的是，一项临床试验显示，阿达格西布和西妥昔单抗联合治疗可显着提高携带KRAS-G12C的CRC患者的缓解率（从19%提高到46%），这表明这种联合治疗可能是该患者人群的有力治疗工具

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. As over 50% of patients with CRC did not show marked responses to the combination therapy, further investigation is required to uncover other potential resistance mechanisms in those cases.

由于超过50%的CRC患者对联合治疗没有明显反应，因此需要进一步调查以发现这些病例中其他潜在的耐药机制。

In this study, we evaluated the resistance mechanisms to KRAS inhibitors by establishing patient-derived cells (PDCs) harboring KRAS-G12C and -G12D mutations in CRC. Through inhibitor library screening and genomic profiling, KRAS-mutated CRC-PDCs were characterized, at least, into two groups from the aspect of intrinsic resistance mechanisms.

在这项研究中，我们通过建立CRC中携带KRAS-G12C和-G12D突变的患者来源细胞（pDC），评估了对KRAS抑制剂的耐药机制。通过抑制剂文库筛选和基因组分析，KRAS突变的CRC pDC至少从内在耐药机制方面分为两组。

More than half of PDCs showed EGFR-mediated MAPK feedback reactivation in the presence of the KRAS inhibitor (first group), and the PI3K pathway activation conferred resistance to the KRAS inhibitor (second group). Pharmacological and immunoblot analyses revealed that one PDC named JC261 in the latter group had Her2.

超过一半的pDC在KRAS抑制剂（第一组）存在下显示EGFR介导的MAPK反馈再激活，并且PI3K途径激活赋予对KRAS抑制剂（第二组）的抗性。药理学和免疫印迹分析显示，后一组中一个名为JC261的PDC具有Her2。

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and PIK3CA

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K111E

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mutation in addition to the KRAS-G12C mutation. PI3K pathway hyperactivation was observed in JC261 cells, with the dual inhibition of PI3K and mTOR effectively suppressing JC261 cell growth. Furthermore, to uncover whether KRAS has oncogenic activity adjacent to the plasma membrane in the PI3K-activating mutation with Her2 amplification harboring cells, we examined KRAS localization.

除KRAS-G12C突变外，还有突变。在JC261细胞中观察到PI3K途径过度活化，PI3K和mTOR的双重抑制有效抑制了JC261细胞的生长。此外，为了揭示KRAS在具有Her2扩增的细胞的PI3K激活突变中是否具有与质膜相邻的致癌活性，我们检查了KRAS的定位。

Immunofluorescence staining of KRAS revealed its localization at the cytoplasm in Her2 amplified with PIK3CA-activated cells, whereas this localization shifted to the plasma membrane in Her2 knockout PI3K low-dependency cells. In addition, Her2 knockout cells recovered KRAS–MAPK activity and were sensitive to sotorasib.

。此外，Her2基因敲除细胞恢复了KRAS-MAPK活性，并且对索托拉西布敏感。

Our findings highlight that Her2 amplification-mediated aberrant KRAS localization may regulate KRAS–MAPK dependency, indicating intrinsic resistance to KRAS inhibitors..

我们的发现强调，Her2扩增介导的异常KRAS定位可能调节KRAS-MAPK依赖性，表明对KRAS抑制剂具有内在抗性。。

Results

结果

KRAS-mutated CRC-PDCs were not solely dependent on KRAS

KRAS突变的CRC pDC不仅仅依赖于KRAS

To uncover the intrinsic or acquired resistance mechanisms to KRAS inhibitors, we established new KRAS G12C- and G12D-positive PDCs from surgically resected specimens of patients with CRC, focusing on the analysis of genomic alterations and drug sensitivity profiles. The cell viability of multiple KRAS-mutated cancer cells was evaluated to examine whether the growth and survival of KRAS-mutated CRC cells depend on mutant KRAS, and PDCs were assessed by treating with the KRAS-G12C inhibitor (sotorasib) or KRAS-G12D inhibitor (MRTX1133).

为了揭示对KRAS抑制剂的内在或获得性耐药机制，我们从CRC患者的手术切除标本中建立了新的KRAS G12C和G12D阳性pDC，重点分析了基因组改变和药物敏感性谱。评估多个KRAS突变的癌细胞的细胞活力以检查KRAS突变的CRC细胞的生长和存活是否依赖于突变的KRAS，并且通过用KRAS-G12C抑制剂（sotorasib）或KRAS-G12D抑制剂（MRTX1133）处理来评估pDC。

In H358, KRAS G12C-positive NSCLC cells, and SW1463, KRAS G12C-mutated CRC cells, 1 µM of sotorasib resulted in a 20% reduction in cell viability compared to the control (Fig. .

在H358，KRAS G12C阳性NSCLC细胞和SW1463，KRAS G12C突变的CRC细胞中，与对照组相比，1µM的sotorasib导致细胞活力降低20%（图）。

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). While SW837, KRAS G12C-mutated CRC cells exhibited intermediate sensitivity to sotorasib, KRAS-G12C PDCs demonstrated insensitivity. In the KRAS-G12D models, commercially available CRC (GP2d cells) were highly sensitive to MRTX1133 (Fig.

)。虽然SW837，KRAS G12C突变的CRC细胞对索托拉西布表现出中等敏感性，但KRAS-G12C pDC表现出不敏感性。。

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and Supplementary Fig.

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). While LS180 CRC and AsPC-1 PDAC cells presented moderate sensitivity to MRTX1133, KRAS-G12D-mutated PDCs demonstrated resistance. Active RAS pull-down assay and immunoblot analysis were then performed to investigate the effect of KRAS inhibitors on these KRAS-mutated CRC-PDCs. The KRAS inhibitor treatment completely suppressed KRAS-GTP for 48 h, whereas suppression of the p-ERK level was temporary and time-dependently recovered until 48 h (Fig.

)。虽然LS180 CRC和AsPC-1 PDAC细胞对MRTX1133表现出中等敏感性，但KRAS-G12D突变的pDC表现出抗性。然后进行活性RAS下拉测定和免疫印迹分析，以研究KRAS抑制剂对这些KRAS突变的CRC pDC的影响。KRAS抑制剂治疗完全抑制KRAS-GTP 48小时，而抑制p-ERK水平是暂时的，并且时间依赖性地恢复到48小时（图）。

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). Furthermore, all PDCs sustained the phospho-AKT levels for 48 h following KRAS inhibitor treatment. Cell viability assay was performed by knocking down KRAS using si-RNAs to examine whether PDCs’ survival depends solely on KRAS. Compared with SW1463, all PDCs tended to maintain cell viability of over 50% following KRAS knockdown, indicating the nonexclusive dependence of KRAS-mutated PDCs on KRAS.

)。此外，在KRAS抑制剂治疗后，所有pDC均维持磷酸化AKT水平48小时。通过使用si-RNA敲低KRAS来进行细胞活力测定，以检查pDC的存活是否仅依赖于KRAS。与SW1463相比，所有pDC在KRAS敲低后倾向于维持超过50%的细胞活力，表明KRAS突变的pDC对KRAS的非排他性依赖性。

These results implied that a single agent of KRAS inhibitors may fail to inhibit cell viability completely in CRC-PDCs similar to the observation of KRAS inhibitor ineffectiveness in clinical trials involving patients with KRAS-mutated CRC, suggesting the activation of a compensatory cell survival pathway upon KRAS suppression..

这些结果表明，单一KRAS抑制剂可能无法完全抑制CRC pDC中的细胞活力，类似于在涉及KRAS突变CRC患者的临床试验中观察到KRAS抑制剂无效，表明KRAS抑制后补偿性细胞存活途径的激活。。

Fig. 1: Single inhibition of mutant KRAS was not sufficient to abrogate cell viability in CRC-PDCs.

图1：突变KRAS的单一抑制不足以消除CRC pDC中的细胞活力。

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Evaluation of sensitivity to KRAS inhibitors. Cells were treated with 1 µM of sotorasib or 30 nM of MRTX1133 for 72 h. Cell viability was detected by CellTiter-Glo assay, and the relative cell viability to the non-treated condition was calculated. Data were expressed as mean ± SD.

评估对KRAS抑制剂的敏感性。用1μMSotorasib或30nM MRTX1133处理细胞72小时。通过CellTiter-Glo测定法检测细胞活力，并计算与未处理条件的相对细胞活力。数据表示为平均值±SD。

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Confirmation of the KRAS downstream signal in KRAS inhibitor treatment. KRAS-G12C and KRAS-G12D CRC cell lines were exposed to 1 µM of sotorasib or 100 nM of MRTX1133 treatment. The cell lysates were collected at each time point and immunoblotted to detect the indicated antibodies (left).

在KRAS抑制剂治疗中确认KRAS下游信号。将KRAS-G12C和KRAS-G12D CRC细胞系暴露于1μMSotorasib或100nM MRTX1133处理。在每个时间点收集细胞裂解物并进行免疫印迹以检测所示抗体（左）。

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Evaluation of KRAS dependency in KRAS-mutated PDCs. The cell viability was measured by CellTiter-Glo assay, and the relative cell viability to si-ctrl cells was calculated. Data were expressed as mean ± SD.

评估KRAS突变的pDC中的KRAS依赖性。通过CellTiter-Glo测定法测量细胞活力，并计算与si-ctrl细胞的相对细胞活力。数据表示为平均值±SD。

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KRAS-G12C with EGFR inhibition proved effective in about half of KRAS-mutated CRC-PDCs

具有EGFR抑制作用的KRAS-G12C在大约一半的KRAS突变的CRC pDC中被证明是有效的

To uncover potential signaling pathways supporting PDC survival upon KRAS inhibition, drug screening was performed using a library of 91 targeted drugs in the presence of a KRAS inhibitor. Inhibitor screening revealed that EGF receptor family inhibitors with the KRAS inhibitor significantly suppressed cell viability in more than half of the CRC-PDCs (Fig.

为了揭示支持KRAS抑制后PDC存活的潜在信号传导途径，在KRAS抑制剂存在下使用91种靶向药物的文库进行药物筛选。抑制剂筛选显示，EGF受体家族抑制剂与KRAS抑制剂显着抑制了超过一半的CRC pDC的细胞活力（图）。

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). Next, we assessed cell viability following treatment with a combination of the KRAS inhibitor and the pan-Erbb family inhibitor afatinib. Both JC288 (KRAS-G12C) and JC117 (KRAS-G12D) CRC-PDCs demonstrated sensitivity to the KRAS inhibitor when treated with 10–30 nM of afatinib in combination (Fig.

)。接下来，我们评估了用KRAS抑制剂和泛Erbb家族抑制剂阿法替尼联合治疗后的细胞活力。当用10-30nM阿法替尼联合治疗时，JC288（KRAS-G12C）和JC117（KRAS-G12D）CRC pDC均显示出对KRAS抑制剂的敏感性（图）。

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b, c and Supplementary Fig.

b、 c和补充图。

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). Immunoblot analysis of the KRAS inhibitor with or without afatinib treatment for JC288 or JC117 revealed that single treatment with KRAS inhibitor induced temporal p-ERK suppression that recovered within 24–48 h, but the combination treatment induced continuous p-ERK suppression for 48 h (Fig.

)。对有或没有阿法替尼治疗JC288或JC117的KRAS抑制剂的免疫印迹分析显示，用KRAS抑制剂单次治疗诱导的时间p-ERK抑制在24-48小时内恢复，但联合治疗诱导持续p-ERK抑制48小时（图1）。

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d, e). Cleaved PARP, an apoptosis marker, was observed to have strongly accumulated in the combination treatment. These results corroborated previously reported findings that MAPK signaling shutdown induced feedback reactivation of the MAPK pathway through EGFR activation in the KRAS-G12C CRC model or BRAF-mutated CRC model as an intrinsic/adaptive resistance mechanism to RAS or RAF inhibitors.

d、 e）。观察到切割的PARP（一种凋亡标志物）在联合治疗中强烈积累。这些结果证实了先前报道的发现，即MAPK信号传导关闭通过KRAS-G12C CRC模型或BRAF突变的CRC模型中的EGFR激活诱导MAPK途径的反馈再激活，作为对RAS或RAF抑制剂的内在/适应性抗性机制。

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. Thus, combining RAS or RAF with EGFR inhibitors, mainly anti-EGFR antibodies, has been reported and clinically applied to overcome resistance in patients with CRC harboring KRAS or BRAF mutations.

因此，已经报道了将RAS或RAF与EGFR抑制剂（主要是抗EGFR抗体）结合使用，并在临床上用于克服携带KRAS或BRAF突变的CRC患者的耐药性。

Fig. 2: Mutant KRAS and EGFR inhibition markedly suppressed cell viability in CRC-PDCs.

图2：突变KRAS和EGFR抑制显着抑制CRC pDC中的细胞活力。

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Inhibitor library screening of KRAS-mutated PDCs. Cells were co-cultured with the indicated inhibitors (bottom), 1 µM of sotorasib or 100 nM of MRTX1133 for 72 h. Cell viability was detected by CellTiter-Glo assay, and relative cell viability to a single treatment of KRAS inhibitors was calculated.

KRAS突变pDC的抑制剂文库筛选。将细胞与指定的抑制剂共培养（底部），1μMSotorasib或100 nM MRTX1133持续72小时。通过CellTiter-Glo测定法检测细胞活力，并计算单次处理KRAS抑制剂的相对细胞活力。

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Combination efficacy of KRAS-G12C inhibitor (sotorasib) plus Erbb family inhibitor (afatinib). KRAS-G12C PDC (JC288) was exposed to 1 µM of sotorasib with or without the indicated concentrations of afatinib for 72 h. Cell viability was detected by CellTiter-Glo assay, and relative cell viability to non-treated condition was calculated.

KRAS-G12C抑制剂（索托拉西布）加Erbb家族抑制剂（阿法替尼）的联合疗效。将KRAS-G12C PDC（JC288）暴露于1msotorasib中，加入或不加入指定浓度的阿法替尼72 h、 通过CellTiter-Glo测定法检测细胞活力，并计算与未处理条件的相对细胞活力。

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Combination efficacy of the KRAS-G12D inhibitor (MRTX1133) plus afatinib. KRAS-G12D PDC (JC117) was exposed to 100 nM of MRTX1133 with or without the indicated concentrations of afatinib for 72 h. Cell viability was detected by CellTiter-Glo assay, and relative cell viability to non-treated conditions was measured.

KRAS-G12D抑制剂（MRTX1133）加阿法替尼的联合疗效。将KRAS-G12D PDC（JC117）暴露于100 nM的MRTX1133中，加入或不加入指定浓度的阿法替尼72 h、 通过CellTiter-Glo测定法检测细胞活力，并测量与未处理条件的相对细胞活力。

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Immunoblotting analysis of the KRAS inhibitor plus afatinib. JC288 and JC117 cells were treated with sotorasib or MRTX1133 for the indicated time. Cell lysates were immunoblotted to detect the indicated antibodies (left).

KRAS抑制剂加阿法替尼的免疫印迹分析。用sotorasib或MRTX1133处理JC288和JC117细胞指定的时间。对细胞裂解物进行免疫印迹以检测所示抗体（左）。

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Therapeutic efficacy of KRAS inhibitor with EGFR antibody in a 3D co-culture system and in vivo

KRAS抑制剂与EGFR抗体在3D共培养系统和体内的治疗效果

Regarding feedback adaptation reports, we hypothesized that our PDCs could also be characterized into an EGFR feedback group. To explore the efficacy of combining the anti-EGFR antibody (cetuximab) with the KRAS-G12C inhibitor (sotorasib) beyond 2D culture conditions, we assessed cell viability using a 3D co-culture model that mimics tumor tissue with stromal tissues and cancer cells layered as well as in vivo xenograft models.

关于反馈适应报告，我们假设我们的pDC也可以表征为EGFR反馈组。为了探索在2D培养条件下将抗EGFR抗体（西妥昔单抗）与KRAS-G12C抑制剂（索托拉西布）组合的功效，我们使用3D共培养模型评估了细胞活力，该模型模拟肿瘤组织与基质组织和癌细胞分层以及体内异种移植模型。

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21

. In the PDC (JC288) partially sensitive to sotorasib, a single treatment with sotorasib was ineffective, but sotorasib with cetuximab treatment markedly inhibited cell viability in the 3D coculture model (Fig.

在对sotorasib部分敏感的PDC（JC288）中，sotorasib的单一治疗无效，但sotorasib与西妥昔单抗治疗显着抑制了3D共培养模型中的细胞活力（图）。

3a

3a级

). We then evaluated the antitumor efficacy of sotorasib-combined cetuximab in a PDC-xenograft model. Compared with the single sotorasib treatment, the combination therapy significantly suppressed tumor growth while preserving body weight (Fig.

)。然后，我们评估了索托拉西布联合西妥昔单抗在PDC异种移植模型中的抗肿瘤功效。与单一索托拉西治疗相比，联合治疗在保持体重的同时显着抑制肿瘤生长（图）。

3b

). These results indicated that the 3D co-culture system showed better recapitulation of in vivo drug efficacy than the 2D culture model, suggesting that its utilization may present a better prediction of in vivo drug efficacy. In addition, we confirmed that feedback reactivation via EGFR was the main cause of the KRAS inhibitor’s intrinsic resistance mechanism in PDCs..

)。这些结果表明，3D共培养系统显示出比2D培养模型更好的体内药物功效重现，表明其利用可能更好地预测体内药物功效。此外，我们证实，通过EGFR的反馈再激活是KRAS抑制剂在pDC中内在耐药机制的主要原因。。

Fig. 3: KRAS-G12C inhibitor with cetuximab combination showed promising effects in PDCs in 3D and in vivo experiments.

图3：具有西妥昔单抗组合的KRAS-G12C抑制剂在3D和体内实验中在pDC中显示出有希望的作用。

a

一

Evaluation of sotorasib plus cetuximab combination efficacy in a 3D culture.

在3D培养中评估索托拉西布联合西妥昔单抗的联合疗效。

b

b类

JC288 cells were subcutaneously transplanted into BALB/c nu/nu mice. Once the average tumor volume reached approximately 150 mm

将JC288细胞皮下移植到BALB/c nu/nu小鼠中。一旦平均肿瘤体积达到约150毫米

3

3

, the mice (

，老鼠(

N

N

= 4) were treated once daily with sotorasib (100 mg/Kg), or sotorasib plus cetuximab (1 mg/body) for 5 days/week. Data were expressed as the mean ± SD, and the Mann–Whitney

。数据表示为平均SD和Mann-Whitney

U

U

-test was used between the control group and the sotorasib plus cetuximab group.

-在对照组和索托拉西布加西妥昔单抗组之间使用测试。

Full size image

全尺寸图像

Genomic profiling revealed PI3K mutation and Her2 amplification in the PDCs resistant to KRAS–EGFR inhibition

基因组分析显示，抗KRAS-EGFR抑制的pDC中PI3K突变和Her2扩增

To identify other resistance mechanisms to KRAS inhibitors besides EGFR reactivation and new therapeutic targets, we focused on PDCs exhibiting high resistance to KRAS–EGFR inhibitor combination therapy. Among these resistant PDCs, JC261 showed marked resistance to the combination therapy, both in vitro and in vivo (Supplementary Fig.

为了确定除EGFR再激活和新的治疗靶点外，对KRAS抑制剂的其他耐药机制，我们专注于对KRAS-EGFR抑制剂联合治疗表现出高耐药性的pDC。在这些耐药pDC中，JC261在体外和体内均显示出对联合治疗的显着抗性（Supplementary Fig.）。

.

.

2a

2a级

and

和

2c

). Despite harboring KRAS

)。

G12C

G12C级

mutation, JC261 seemed to present exclusive independence on KRAS for its viability (Fig.

突变，JC261似乎对KRAS的生存能力具有排他性的独立性（图）。

1

1

a and

a和

d

d

). Therefore, to investigate the genomic aberration in the KRAS-mutated PDCs, target sequencing analysis was performed using 12 surgically resected CRC tumors and normal paired samples with KRAS-G12C or -G12D mutations (Fig.

)。因此，为了研究KRAS突变的pDC中的基因组畸变，使用12个手术切除的CRC肿瘤和具有KRAS-G12C或-G12D突变的正常配对样品进行靶标测序分析（图）。

4a

4a级

).

).

APC

APC公司

and

和

TP53

TP53型

mutations were found in 83% and 67% of all samples, respectively. Following these genes, the

在所有样品中分别有83%和67%发现突变。遵循这些基因

PIK3CA

PIK3CA

mutation was the third most frequently mutated in KRAS-mutated CRC samples, including JC261. To gain deeper insights into the genetic alterations, including gene amplification in JC261, target-sequencing data were analyzed. Copy number variation analysis revealed

突变是KRAS突变的CRC样本（包括JC261）中突变频率第三高的突变。为了更深入地了解遗传改变，包括JC261中的基因扩增，分析了靶标测序数据。拷贝数变异分析显示

ERBB2

ERBB2

amplification on chromosome 17 (Fig.

17号染色体上的扩增（图）。

4b

4b级

and Supplementary Fig.

。

3a

3a级

,

,

3b

3b级

). Compared with the normal sample,

)。与正常样品相比，

Erbb2

Erbb2

was 16-fold amplified. DNA sequencing analysis identified

。鉴定出DNA测序分析

APC

APC公司

,

,

TP53

TP53型

,

,

KRAS

克拉斯

, and

，以及

ERBB2

ERBB2

gene alterations in JC261. Furthermore, immunoblot analysis was performed to confirm the expression and activation of molecules in the Her2 and PI3K signaling pathways. Extremely high expression levels of total Her2 and phosphorylated Her2, and relatively high level of phosphorylated AKT were detected in JC261 cells compared with the other KRAS-mutated PDCs (Fig.

JC261中的基因改变。此外，进行免疫印迹分析以确认Her2和PI3K信号传导途径中分子的表达和活化。与其他KRAS突变的pDC相比，在JC261细胞中检测到总Her2和磷酸化Her2的极高表达水平以及相对较高水平的磷酸化AKT（图）。

.

.

4c

4c级

). These data suggested that Her2 amplification and PIK3CA mutation primarily contributed to resistance to sotorasib through the continuous activation of Her2-PI3K signaling in JC261.

)。这些数据表明，Her2扩增和PIK3CA突变主要通过JC261中Her2-PI3K信号的连续激活而导致对索托拉西的抗性。

Fig. 4: Her2-PI3K pathway gene alterations were detected in KRAS-G12C inhibitor plus cetuximab combination-resistant PDCs.

图4：在KRAS-G12C抑制剂加西妥昔单抗联合耐药pDC中检测到Her2-PI3K途径基因改变。

a

一

Genomic profiling of KRAS-mutated PDCs. Genomic DNA (gDNA) was purified from surgically resected clinical samples, and NGS was executed using 108 cancer-related gene-focused libraries. OncoPlot revealed alterations of the indicated gene (left) in each sample (bottom).

KRAS突变的pDC的基因组分析。从手术切除的临床样品中纯化基因组DNA（gDNA），并使用108个癌症相关基因聚焦文库执行NGS。OncoPlot显示每个样品（底部）中指定基因（左）的改变。

b

b类

CNV alterations of JC261 cells. The copy ratio was analyzed by CNVkit using target re-sequencing data in Fig. 4a and visualized in the

JC261细胞的CNV改变。拷贝比由CNVkit使用图4a中的靶标重测序数据进行分析，并在

ERBB2

ERBB2

region.

地区。

c

c级

Confirmation of Her2 expression and PI3K signaling. Cell lysates were immunoblotted to detect the indicated antibodies (left).

确认Her2表达和PI3K信号传导。对细胞裂解物进行免疫印迹以检测所示抗体（左）。

Full size image

全尺寸图像

Triple inhibition of PI3K, mTOR, and KRAS-G12C robustly induced apoptosis in Her2

PI3K，mTOR和KRAS-G12C的三重抑制强烈诱导Her2细胞凋亡

amp

安培

, PI3K, and KRAS-mutated-positive PDCs

，PI3K和KRAS突变的阳性pDC

In addition to genomic profiling, inhibitor library screening of JC261 revealed its vulnerability to PI3K and mTOR inhibition by BEZ235 (PI3K/mTOR dual inhibitor). Therefore, the efficacy of PI3K and/or mTOR inhibition against the cell viability of JC261 was investigated. To inhibit PI3K and/or mTOR, the cell viability of JC261 cells was evaluated by treatment with BEZ235, GDC0941, a PI3K inhibitor, and PP242, an mTOR inhibitor.

除了基因组分析外，JC261的抑制剂文库筛选还揭示了其对BEZ235（PI3K/mTOR双重抑制剂）抑制PI3K和mTOR的脆弱性。因此，研究了PI3K和/或mTOR抑制对JC261细胞活力的功效。为了抑制PI3K和/或mTOR，通过用PI3K抑制剂BEZ235，GDC0941和mTOR抑制剂PP242处理来评估JC261细胞的细胞活力。

Pharmacological analysis revealed that a single treatment with BEZ235 or GDC0941 plus PP242 combination significantly reduced cell viability (Fig. .

药理学分析显示，用BEZ235或GDC0941加PP242联合治疗可显着降低细胞活力（图）。

5a

5a级

). Furthermore, adding sotorasib to BEZ235 or GDC0941 in combination with PP242 intensively inhibited the cell viability of JC261 cells. Next, we assessed cell viability by PIK3CA-specific silencing with/without mTOR inhibition. The cell viability assay indicated that PIK3CA silencing and mTOR inhibition markedly suppressed cell viability more than single PIK3CA silencing in JC261, but not in JC288 (Fig.

)。此外，将sotorasib加入BEZ235或GDC0941与PP242组合强烈抑制JC261细胞的细胞活力。接下来，我们通过有/没有mTOR抑制的PIK3CA特异性沉默评估细胞活力。细胞活力测定表明，PIK3CA沉默和mTOR抑制比JC261中的单个PIK3CA沉默显着抑制细胞活力，但在JC288中没有（图）。

.

.

5b

5b条

and Supplementary Fig.

。

4a

4a级

). To confirm the PI3K signaling alterations in the PI3K pathway inhibitor treatment, we performed immunoblot analysis. The single treatment of GDC0941 or PP242 resulted in a 24-h abrogation of p-AKT and p-S6 levels but failed to induce PARP cleavage (Fig.

)。为了证实PI3K途径抑制剂治疗中的PI3K信号改变，我们进行了免疫印迹分析。GDC0941或PP242的单一处理导致p-AKT和p-S6水平的24小时消除，但未能诱导PARP裂解（图）。

5c

5摄氏度

). In contrast, dual inhibition of PI3K and mTOR resulted in decreased p-AKT and p-S6 levels and the accumulation of PARP cleavage. In addition, triple inhibition of PI3K, mTOR, and KRAS-G12C suppressed p-AKT and pS6 levels, leading to a greater suppression of p-mTOR and accumulation of cleaved PARP than dual inhibition of PI3K and mTOR.

)。相反，PI3K和mTOR的双重抑制导致p-AKT和p-S6水平降低以及PARP裂解的积累。此外，PI3K，mTOR和KRAS-G12C的三重抑制抑制了p-AKT和pS6水平，导致p-mTOR的抑制和切割的PARP的积累比PI3K和mTOR的双重抑制更大。

These results indicated that dual inhibition of PI3K and mTOR is necessary to suppress cell viability, but incorporating sotorasib treatment can induce robust apoptosis. Consistently, cell viability of JC261 was significantly suppressed by PI3K and mTOR inhibition, and additive growth suppression was observed by adding sotorasib, indicating that PIK3CA and mTOR inhibition with KRAS-G12C inhibition significantly suppressed cell viability more than PI3K and mTOR inhibition in JC261 (Fig.

这些结果表明，PI3K和mTOR的双重抑制对于抑制细胞活力是必需的，但是掺入索托拉西布治疗可以诱导强烈的细胞凋亡。一致地，PI3K和mTOR抑制显着抑制了JC261的细胞活力，并且通过添加sotorasib观察到加性生长抑制，表明PIK3CA和mTOR抑制与KRAS-G12C抑制相比，JC261中的PI3K和mTOR抑制显着抑制了细胞活力（图。

.

.

5d

5天

).

).

Fig. 5: Efficacy of dual inhibition of PI3K and mTOR in PIK3CA, ERBB2-amplified KRAS-G12C PDC.

。

a

一

Pharmacological analysis of PI3K pathway inhibitors in JC261 cells. The cells were treated with the indicated 1 µM inhibitors with or without 1 µM of sotorasib for 72 h. Cell viability was analyzed by CellTiter-Glo assay, and the relative cell viability to DMSO treatment was calculated. Data were shown as mean ± SD.

JC261细胞中PI3K途径抑制剂的药理学分析。用指定的1m抑制剂处理细胞，加入或不加入1m的索托拉西布72 h、 通过CellTiter-Glo测定分析细胞活力，并计算与DMSO处理的相对细胞活力。数据显示为平均值±SD。

.

.

b

b类

Cell viability analysis of PI3K knockdown and mTOR inhibition. 20 nM of each siRNA was introduced to cells for 48 h. The cell viability was analyzed using the CellTiter-Glo assay, and the relative cell viability to si-control treated condition was calculated. Data were shown as mean ± SD.

PI3K敲低和mTOR抑制的细胞活力分析。将20nm的每种siRNA引入细胞48小时。使用CellTiter-Glo测定法分析细胞活力，并计算相对于si对照处理条件的相对细胞活力。数据显示为平均值±SD。

c

c级

Immunoblotting analysis of PI3K, mTOR, and KRAS inhibition. JC261 cells were incubated with the indicated inhibitors. Cell lysates were collected at each time point and immunoblotted to detect the indicated antibodies (left).

PI3K，mTOR和KRAS抑制的免疫印迹分析。将JC261细胞与指定的抑制剂一起温育。在每个时间点收集细胞裂解物并进行免疫印迹以检测所示抗体（左）。

d

d

Sensitivity to BEZ235 with or without sotorasib (upper) or PP242 with or without GDC0941 and sotorasib in JC261 cells. The cells were treated with the serially diluted BEZ235 or PP242 and the indicated concentration of GDC0941 and sotorasib for 3 days.

在JC261细胞中，对有或没有sotorasib（上）的BEZ235或有或没有GDC0941和sotorasib的PP242的敏感性。用连续稀释的BEZ235或PP242和指定浓度的GDC0941和sotorasib处理细胞3天。

Full size image

全尺寸图像

Her2 amplification conferred resistance to the KRAS inhibitor

Her2扩增赋予对KRAS抑制剂的抗性

Then, the mechanisms underlying JC261 cells’ dependence on the PI3K pathway rather than on the MAPK pathway was investigated, although JC261 harbors

然后，研究了JC261细胞依赖PI3K途径而不是MAPK途径的潜在机制，尽管JC261具有

KRAS

克拉斯

G12C

G12C级

mutation. Immunoblot analysis revealed that JC261 had a higher relative signal intensity of p-AKT/total AKT level than PIK3CA

突变。免疫印迹分析显示，JC261的p-AKT/总AKT水平的相对信号强度高于PIK3CA

H1047R

H1047R

-mutated KRAS

-突变的KRAS

G12C

G12C级

-positive PDC (JC312-2) (Fig.

-阳性PDC（JC312-2）（图）。

4c

4c级

). Moreover, previous studies indicated that PIK3CA

)。此外，先前的研究表明PIK3CA

K111E

K111E

mutation activated p-AKT more significantly than PIK3CA

突变激活p-AKT比PIK3CA更显著

WT

重量

in USO2 osteosarcoma cell models

在USO2骨肉瘤细胞模型中

22

22

. However, p-AKT levels were not as elevated as those in PIK3CA

然而，p-AKT水平并不像PIK3CA那样升高

H1047R

H1047R

(a well-known hotspot mutation). Collectively, Her2 amplification may have contributed to high PI3K axis activation and the dependency of JC261 cell viability on PI3K/mTOR. Therefore, we introduced two individual guide RNAs (gRNA) and established Her2 knockout JC261 cell lines (Her2 KO#1, Her2 KO#3) (Fig.

（一个众所周知的热点突变）。总的来说，Her2扩增可能有助于高PI3K轴活化和JC261细胞活力对PI3K/mTOR的依赖性。因此，我们引入了两个单独的指导RNA（gRNA）并建立了Her2敲除JC261细胞系（Her2 KO#1，Her2 KO#3）（图）。

.

.

6a

6a

). Immunoblot analysis revealed that Her2 KO resulted in reduced total and phosphorylated Her2 expression while suppressing p-Her3 and p-AKT levels in both Her2 KO cells (Fig.

)。免疫印迹分析显示，Her2 KO导致Her2总表达和磷酸化表达降低，同时抑制Her2 KO细胞中的p-Her3和p-AKT水平（图）。

6b

6b条

). In contrast, p-ERK levels were upregulated in Her2 KO cells compared with parental JC261 cells. These results suggest that Her2 KO induced Her2-Her3-PI3K pathway downregulation and p-ERK activation. To clarify whether p-ERK upregulation was elicited by KRAS activation, we assessed the amount of GTP form of KRAS in parent and Her2 KO cells.

)。相反，与亲本JC261细胞相比，Her2 KO细胞中的p-ERK水平上调。这些结果表明Her2 KO诱导Her2-Her3-PI3K途径下调和p-ERK活化。为了阐明KRAS激活是否引起p-ERK上调，我们评估了亲本和Her2 KO细胞中KRAS的GTP形式的量。

Active RAS pull-down assay demonstrated that GTP-KRAS was slightly upregulated in Her2 knockout cells, and completely suppressed by sotorasib treatment for 3 to 24 hr (Fig. .

活性RAS下拉试验表明，GTP-KRAS在Her2基因敲除细胞中略有上调，并被索托拉西布处理3至24小时完全抑制（图）。

6

6

b and

b和

c

c级

). As the KRAS–MAPK pathway seemed to be activated in Her2 knockout cells, we evaluated their sensitivity to sotorasib. Long-term cell proliferation assay showed that Her2 knockout cells recovered sensitivity to sotorasib (Fig.

)。由于KRAS-MAPK途径似乎在Her2基因敲除细胞中被激活，我们评估了它们对索托拉西布的敏感性。长期细胞增殖试验表明，Her2基因敲除细胞恢复了对索托拉西布的敏感性（图）。

6d

6天

and Supplementary Fig

和补充图

5

5

). In addition, Her2 knockout cells were more sensitive to MEK inhibitor trametinib than parental JC261 cells (Fig.

)。此外，Her2敲除细胞比亲本JC261细胞对MEK抑制剂曲美替尼更敏感（图）。

6e

6e型

). These data indicated that the PI3K dependency and resistance to the KRAS inhibitor was mainly attributed to

)。这些数据表明，PI3K依赖性和对KRAS抑制剂的耐药性主要归因于

Her2

Her2

amplification in JC261 and that the dependency of the survival pathway transferred to the KRAS–MAPK pathway by Her2 knockout.

JC261中的扩增，并且存活途径的依赖性通过Her2敲除转移到KRAS-MAPK途径。

Fig. 6: Her2 knockout induced suppression of the Her2-Her3-PI3K pathway and recovered sensitivity to sotorasib.

。

a

一

Certification of Cas9-mediated Her2 knockout. JC261 cells transduced sgHer2 and established Her2 knockout (KO) cell lines. Cell lysates were collected to perform immunoblotting using the indicated antibodies.

Cas9介导的Her2敲除的认证。JC261细胞转导sgHer2并建立Her2敲除（KO）细胞系。收集细胞裂解物以使用指定的抗体进行免疫印迹。

b

b类

Comparison of KRAS downstream signaling between JC261 parent cells and Her2 KO cells in KRAS-G12C inhibition. Cells were exposed to 1 µM of sotorasib each time; cell lysates were then collected and immunoblotted to detect the indicated antibodies.

在KRAS-G12C抑制中比较JC261亲本细胞和Her2 KO细胞之间的KRAS下游信号传导。每次将细胞暴露于1μMSotorasib；然后收集细胞裂解物并进行免疫印迹以检测所示抗体。

c

c级

Evaluation of GTP-KRAS by Ras pull-down assay. Cell lysates were collected, and Ras pull-down assay was performed to detect GTP-KRAS.

通过Ras下拉测定评估GTP-KRAS。收集细胞裂解物，并进行Ras下拉测定以检测GTP-KRAS。

d

d

Sensitivity to sotorasib in Her2 KO cells. The cells were treated with each concentration of sotorasib for 6 days.

Her2 KO细胞对sotorasib的敏感性。用每种浓度的索托拉西布处理细胞6天。

e

e

Sensitivity to trametinib in Her2 KO cells. The cells were treated with each concentration of trametinib for 6 days. Cell viability was calculated by CellTiter-Glo assay, and relative cell viability to nontreatment conditions was calculated. Data were shown as mean ± SD.

Her2 KO细胞对曲美替尼的敏感性。用每种浓度的曲美替尼处理细胞6天。通过CellTiter-Glo测定法计算细胞活力，并计算相对于非处理条件的相对细胞活力。数据显示为平均值±SD。

Full size image

全尺寸图像

KRAS was localized at the cytoplasm in Her2-amplified PDCs

KRAS定位于Her2扩增的pDC的细胞质中

We then sought to determine how the Her2 knockout upregulated the activity of the KRAS–MAPK pathway. As plasma membrane localization of KRAS is crucial for its catalytic activity, we focused on KRAS localization and performed immunofluorescence staining to evaluate intracellular KRAS localization

然后，我们试图确定Her2基因敲除如何上调KRAS-MAPK途径的活性。由于KRAS的质膜定位对其催化活性至关重要，因此我们专注于KRAS的定位，并进行了免疫荧光染色以评估细胞内KRAS的定位

23

23

. Notably, immunofluorescence staining of KRAS in JC261 demonstrated that KRAS was mainly localized at the cytoplasm in parental JC261 cells, while its localization changed to the plasma membrane in Her2 knockout cells (Fig.

值得注意的是，JC261中KRAS的免疫荧光染色表明KRAS主要定位于亲本JC261细胞的细胞质中，而其定位在Her2敲除细胞的质膜上（图）。

7a

7a个

). To confirm whether aberrant KRAS localization was exclusive to JC261 cells, we performed immunofluorescence staining of KRAS in other driver oncogene-positive cell lines. In SW48 CRC cells harboring EGFR-activating mutation, and JC288, a KRAS-G12C-positive PDC, KRAS was observed in the plasma membrane (Fig.

)。。在携带EGFR激活突变的SW48 CRC细胞和KRAS-G12C阳性PDC JC288中，在质膜中观察到KRAS（图）。

.

.

7b

7b条

). In contrast, KRAS was localized at the cytoplasm in JC69 cells, a

)。相反，KRAS位于JC69细胞的细胞质中

Her2

Her2

-amplified KRAS WT PDC. These results suggested that Her2 amplification may trigger aberrant KRAS localization, resulting in low dependency and sensitivity to the KRAS–MAPK pathway.

-扩增的KRAS WT PDC。这些结果表明，Her2扩增可能触发异常的KRAS定位，导致对KRAS-MAPK途径的低依赖性和敏感性。

Fig. 7: KRAS was mainly localized at the cytoplasm in Her2-positive CRC.

图7:KRAS主要位于Her2阳性CRC的细胞质中。

a

一

,

,

b

b类

Immunofluorescence analysis of KRAS in various cell lines. Representative immunofluorescence images are shown for KRAS (red), E-cadherin, p-Her2

各种细胞系中KRAS的免疫荧光分析。显示了KRAS（红色），E-钙粘蛋白，p-Her2的代表性免疫荧光图像

14

14

, and Hoechst (blue). Scale bar: 20 μm.

，和Hoechst（蓝色）。比例尺：20微米。

c

c级

Graphical image of Her2-amplified-mediated KRAS localization in this study.

。

Full size image

全尺寸图像

Discussion

讨论

The mutant KRAS gene has recently become a targetable oncogene, which can be achieved by directly inhibiting KRAS-G12C with several covalent inhibitors or G12D with non-covalent inhibitor(s). In addition, pan-KRAS inhibitors have been developed and are awaiting future clinical validation. However, KRAS.

突变的KRAS基因最近已成为可靶向的癌基因，可以通过用几种共价抑制剂直接抑制KRAS-G12C或用非共价抑制剂直接抑制G12D来实现。此外，泛KRAS抑制剂已经开发出来，正在等待未来的临床验证。然而，克拉斯。

G12C

G12C级

inhibitors’ clinical trials have revealed clinical efficiency variations between patients with KRAS G12C-positive NSCLC and those with CRC. EGFR, PI3K, mTOR, or CDK4/6 have been implicated in adaptive resistance to KRAS inhibitors

抑制剂的临床试验揭示了KRAS G12C阳性NSCLC患者与CRC患者之间的临床效率差异。EGFR，PI3K，mTOR或CDK4/6与KRAS抑制剂的适应性耐药有关

14

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,

,

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,

,

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17

,

,

24

24

. Although co-targeting KRAS inhibitors with these inhibitors resulted in an enhanced antitumor effect in NSCLC or PDAC models, further investigation is needed to understand the resistance mechanisms to KRAS inhibitors in CRC. In this study, we analyzed our original KRAS G12C and G12D-mutated CRC-PDCs to identify the intrinsic resistance mechanisms to KRAS inhibitors.

尽管与这些抑制剂共同靶向KRAS抑制剂在NSCLC或PDAC模型中产生增强的抗肿瘤作用，但需要进一步研究以了解CRC中KRAS抑制剂的耐药机制。在这项研究中，我们分析了我们最初的KRAS G12C和G12D突变的CRC pDC，以确定对KRAS抑制剂的内在耐药机制。

Consequently, we categorized PDCs into MAPK-reactivation and PI3K-dependent groups regarding pathway activation under KRAS inhibitor treatment. We also provided insights into the aberrant localization of KRAS-conferred intrinsic resistance to the KRAS inhibitor in Her2 amplification-mediated PI3K-dependent CRC..

因此，我们将pDC分为MAPK再激活和PI3K依赖性组，涉及KRAS抑制剂治疗下的途径激活。我们还提供了关于在Her2扩增介导的PI3K依赖性CRC中KRAS异常定位赋予KRAS抑制剂内在抗性的见解。。

EGFR-mediated MAPK-reactivation is a common occurrence due to KRAS, BRAF, and MEK inhibitor treatment, resulting in resistance to single MAPK inhibitors in patients with BRAF V600E and KRAS G12C-positive CRC

由于KRAS、BRAF和MEK抑制剂的治疗，EGFR介导的MAPK再激活是一种常见的现象，导致BRAF V600E和KRAS G12C阳性CRC患者对单一MAPK抑制剂产生耐药性

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17

,

,

19

19

,

,

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. To overcome this primary resistance, clinical trials have evaluated combination therapy of the KRAS-G12C inhibitor (adagrasib) and anti-EGFR antibody (cetuximab) in patients with KRAS-G12C CRC, revealing a markedly improved response rate (46%) compared to the limited rate of adagrasib monotherapy (19%).

为了克服这种原发性耐药，临床试验评估了KRAS-G12C抑制剂（阿达格拉西布）和抗EGFR抗体（西妥昔单抗）在KRAS-G12C CRC患者中的联合治疗，显示与阿达格拉西布单药治疗的有限率（19%）相比，显着提高了缓解率（46%）。

18

18

. Notably, we also found here that sotorasib or MRTX1133 monotherapy failed to constantly suppress MAPK and PI3K signaling activity, resulting in limited antitumor efficacy in vitro and in vivo. In contrast, afatinib or cetuximab combination therapy triggered the suppression of MAPK-reactivation and PI3K signaling and decreased cell viability, suggesting that the combination strategy could overcome the intrinsic resistance to the KRAS inhibitor in KRAS-G12C and -G12D models.

值得注意的是，我们在这里还发现，sotorasib或MRTX1133单一疗法未能持续抑制MAPK和PI3K信号传导活性，导致体外和体内抗肿瘤功效有限。相反，阿法替尼或西妥昔单抗联合治疗引发了MAPK再激活和PI3K信号传导的抑制，并降低了细胞活力，这表明该联合策略可以克服KRAS-G12C和-G12D模型中对KRAS抑制剂的内在抗性。

This may also indicate that a combination therapy can prove effective in other KRAS-mutated CRC models, including G13D, G12V, and G12S CRC. In this study, we focused on the combined inhibition of EGFR and mutant KRAS while considering that multiple RTKs can potentially confer resistance to KRAS inhibitors, as previously reported.

这也可能表明联合治疗可以证明在其他KRAS突变的CRC模型中有效，包括G13D，G12V和G12S CRC。在这项研究中，我们专注于EGFR和突变KRAS的联合抑制，同时考虑到多种RTK可能潜在地赋予对KRAS抑制剂的抗性，如先前报道的。

Therefore, to prevent feedback reactivation via RTK activation, SHP2 or SOS1 could be a co-target for CRC treatment.

因此，为了防止通过RTK激活进行反馈再激活，SHP2或SOS1可能是CRC治疗的共同目标。

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.

.

Although adagrasib-combined cetuximab treatment markedly showed favorable results in clinical trials, approximately half of the patients with CRC showed resistance to the combination therapy, and the primary resistance mechanisms remain unknown. In this study, we discovered that

尽管阿达格西布联合西妥昔单抗治疗在临床试验中显示出良好的结果，但大约一半的CRC患者对联合治疗表现出耐药性，主要耐药机制仍然未知。

PIK3CA

PIK3CA

hotspot mutations with

热点突变

ERBB2

ERBB2

amplification could confer intrinsic resistance to combination therapy in CRC models.

扩增可以在CRC模型中赋予对联合治疗的内在抗性。

PIK3CA

PIK3CA

mutations are found in 10–20% of CRC cases but are not mutually exclusive with KRAS mutations in CRC

在10-20%的CRC病例中发现突变，但与CRC中的KRAS突变并不相互排斥

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. KRAS and PIK3CA concomitant mutations were found in 26.7% of CRC cases in the MSK-IMPACT study, although our genomic analysis showed 42% due to the small sample size

在MSK-IMPACT研究中，26.7%的CRC病例发现了KRAS和PIK3CA伴随突变，尽管我们的基因组分析显示42%是由于样本量小

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. In particular, the occurrence of E545K at exon 9 and H1047R at exon 20 is prevalent in cancers, and these mutations can constitutively activate PI3K signaling

特别是，E545K在第9外显子和H1047R在第20外显子的发生在癌症中很普遍，这些突变可以组成性激活PI3K信号传导

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,

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. Previous reports have shown that

以前的报告显示

PIK3CA

PIK3CA

mutations can confer acquired resistance to KRAS G12C inhibitor and KRAS G12C inhibitor–cetuximab combination therapy in patients with NSCLC and CRC

突变可使NSCLC和CRC患者获得对KRAS G12C抑制剂和KRAS G12C抑制剂-西妥昔单抗联合治疗的耐药性

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. We also confirmed that PIK3CA H1047R-mutated KRAS-G12C PDC (JC312-2) presented primary resistance to combination therapy. Although the PIK3CA K111E mutation may induce resistance to combination therapy, the contribution of Her2 amplification seemed to be considerable. PI3K-activating mutations, particularly H1047R, could serve as predictive markers for resistance to both KRAS inhibitor monotherapy and KRAS plus Erbb family inhibitor combination therapy..

我们还证实，PIK3CA H1047R突变的KRAS-G12C PDC（JC312-2）对联合治疗具有主要耐药性。尽管PIK3CA K111E突变可能诱导对联合治疗的耐药性，但Her2扩增的贡献似乎相当大。PI3K激活突变，特别是H1047R，可以作为对KRAS抑制剂单一疗法和KRAS加Erbb家族抑制剂联合疗法耐药的预测标志物。。

In this study, to overcome

在这项研究中，要克服

PIK3CA

PIK3CA

K111E

K111E

and

和

Her2

Her2

amp

安培

-positive KRAS-G12C CRC cells, we discovered that triple inhibition of PI3K, mTOR, and KRAS-G12C markedly suppressed cell viability in the in vitro experiment. However, a previous report uncovered severe toxicity when MEK and PI3K inhibitors were used for treatment in patients with RAS and PIK3CA-mutated CRC.

-阳性KRAS-G12C CRC细胞，我们发现PI3K，mTOR和KRAS-G12C的三重抑制在体外实验中显着抑制细胞活力。然而，先前的报道发现，当MEK和PI3K抑制剂用于治疗RAS和PIK3CA突变的CRC患者时，会产生严重的毒性。

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. Although BEZ235 showed satisfactory antitumor activity against various cancer types in a preclinical model, poor tolerability and high adverse events were observed in clinical trials

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. Thus, the challenges associated with dual PI3K and MAPK pathway inhibition persist in clinical settings. Hence, further research is necessary to explore therapeutic strategies capable of overcoming the activation of both MAPK and PI3K pathways in CRC.

因此，与双重PI3K和MAPK途径抑制相关的挑战在临床环境中仍然存在。因此，有必要进一步研究以探索能够克服CRC中MAPK和PI3K途径激活的治疗策略。

Post-translational modification of KRAS plays an important role in KRAS–MAPK pathway activation. In particular, farnesylation at the position 185 cysteine residue results in KRAS anchoring at the plasma membrane, initiating the activation of KRAS and its downstream signaling

KRAS的翻译后修饰在KRAS–MAPK途径激活中起重要作用。特别是，185位半胱氨酸残基的法尼基化导致KRAS锚定在质膜上，启动KRAS及其下游信号的激活

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. Inhibiting plasma membrane KRAS localization using farnesyltransferase inhibitors suppressed MAPK activity, showing an antitumor effect on cancers driven by KRAS mutations

.使用法尼基转移酶抑制剂抑制质膜KRAS定位抑制了MAPK活性，显示出对KRAS突变驱动的癌症的抗肿瘤作用

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. Intriguingly, we observed that KRAS was mainly localized at the cytoplasm in Her2 amplification with KRAS-G12C mutation-positive cells, and KRAS was localized at the plasma membrane in the Her2 KO cells. However, mRNA expression of farnesyltransferase-related genes remained unchanged between the Her2 amp and Her2 KO cells.

有趣的是，我们观察到KRAS主要位于具有KRAS-G12C突变阳性细胞的Her2扩增的细胞质中，而KRAS位于Her2 KO细胞的质膜上。然而，在Her2 amp和Her2 KO细胞之间，法尼基转移酶相关基因的mRNA表达保持不变。

Thus, Her2 amplification-mediated aberrant KRAS localization may occur in a farnesyltransferase-independent manner. Moreover, Her2 amplification with KRAS WT cells demonstrated cytoplasmic KRAS localization, suggesting that Her2 mainly affected aberrant KRAS localization. KRAS localization remained unaltered following phosphoryrated-Her2 inhibition by pan-Erbb family inhibitor (afatinib) treatment.

因此，Her2扩增介导的异常KRAS定位可能以法尼基转移酶非依赖性方式发生。此外，用KRAS WT细胞进行的Her2扩增显示出细胞质KRAS定位，表明Her2主要影响异常的KRAS定位。泛Erbb家族抑制剂（阿法替尼）抑制磷酸化Her2后，KRAS定位保持不变。

These results indicate that KRAS cytoplasmic localization may not necessarily be elicited via downstream signaling of Her2 and that Her2-interacting or -related scaffold proteins may be involved in blocking KRAS anchoring at the plasma membrane. Other than that, Her2–EGFR driven signaling may evoke the other signaling pathway or effector proteins, contributing the intracellular KRAS localization.

这些结果表明，KRAS细胞质定位可能不一定是通过Her2的下游信号传导引起的，并且Her2相互作用或相关的支架蛋白可能参与阻断KRAS锚定在质膜上。除此之外，Her2-EGFR驱动的信号传导可能会引起其他信号传导途径或效应蛋白，从而促进细胞内KRAS的定位。

According to the changing KRAS localization to the plasma membrane by Her2 KO, we detected the activation of KRAS–MAPK signaling and the downregulation of PI3K signaling. These results suggest that cytoplasmic KRAS localization suppresses KRAS–MAPK pathway activity at low levels in JC261. Furthermore, a JC261 in vivo study demonstrated that sotorasib treatment resulted in a faster tumor growth rate than control treatment.

根据Her2 KO改变KRAS在质膜上的定位，我们检测到KRAS-MAPK信号的激活和PI3K信号的下调。这些结果表明，细胞质KRAS定位在JC261中以低水平抑制KRAS-MAPK途径的活性。此外，JC261体内研究表明，索托拉西布治疗比对照治疗导致更快的肿瘤生长速度。

Because cytoplasmic KRAS localization suppresses KRAS–MAPK pathway activity at low levels, activating the MAPK pathway by mutant KRAS and the PI3K pathway by PIK3CA mutation with Her2 amplification may be harmful due to too much oncogene signali.

。

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45

,

,

46

46

. In this study, we identified the alteration in KRAS localization as a resistance mechanism to KRAS inhibitors. This phenomenon may also be associated with resistance to Her2-targeted therapy in patients with Her2 amplification-positive CRC. In such cases, KRAS is presumed to localize in the cytoplasm in Her2-PI3K dependency while shifting to the plasma membrane when KRAS–MAPK signaling is essential for cell survival during Her2-targeted therapy.

在这项研究中，我们确定了KRAS定位的改变是对KRAS抑制剂的耐药机制。这种现象也可能与Her2扩增阳性CRC患者对Her2靶向治疗的耐药性有关。在这种情况下，当KRAS-MAPK信号传导对于Her2靶向治疗期间的细胞存活至关重要时，推测KRAS定位于Her2-PI3K依赖性的细胞质中，同时转移至质膜。

Thus, KRAS dislocalization may promote resistance to both KRAS inhibitors and other resistance mechanisms..

因此，KRAS脱位可能会促进对KRAS抑制剂和其他耐药机制的耐药性。。

Future studies should investigate how to directly regulate KRAS localization from the plasma membrane to the cytoplasm. Related to this, the intracellular EML4-ALK fusion protein has been shown to recruit RTK adaptor proteins, resulting in the elicitation of cytoplasmic KRAS localization

未来的研究应该研究如何直接调节KRAS从质膜到细胞质的定位。与此相关的是，细胞内EML4-ALK融合蛋白已被证明可以募集RTK衔接蛋白，从而引发细胞质KRAS定位

47

47

. This might suggest that exploring cytoplasmic KRAS-interacting proteins could provide better insights into the regulation of KRAS localization. Recent reports indicate that mislocalization of the Scribble protein leads to YAP-mediated MRAS-MAPK activation, causing adaptive resistance to KRAS-G12C inhibitors.

这可能表明探索细胞质KRAS相互作用蛋白可以更好地了解KRAS定位的调控。最近的报道表明，Scribble蛋白的错误定位导致YAP介导的MRAS-MAPK激活，导致对KRAS-G12C抑制剂的适应性抗性。

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. Overall, considering protein localization in the context of adaptive drug resistance mechanisms is crucial.

总体而言，在适应性耐药机制的背景下考虑蛋白质定位至关重要。

Unfortunately, owing to infrequence in Her2-amplified CRC cases ( ~ 2 or 3% of CRC) and failure to establish Her2-overexpressed KRAS-G12C cells, we could not identify the detailed mechanism of aberrant KRAS localization. In this study, we discovered several KRAS-mutated PDC cell lines showing intrinsic resistance to KRAS inhibitor-combined EGFR inhibitor treatment.

不幸的是，由于Her2扩增的CRC病例不常见（CRC的〜2或3%），并且未能建立Her2过表达的KRAS-G12C细胞，我们无法确定异常KRAS定位的详细机制。在这项研究中，我们发现了几种KRAS突变的PDC细胞系，它们显示出对KRAS抑制剂联合EGFR抑制剂治疗的内在抗性。

Inhibitor screening and genomic profiling revealed Her2 amplification and PI3K mutation in a resistant PDC, as well as vulnerability to KRAS-G12C, PI3K, and mTOR triple inhibition. Furthermore, we emphasize that cytoplasmic KRAS localization driven by Her2 amplification maintains a low dependency on KRAS–MAPK, leading to intrinsic resistance to KRAS inhibitors.

抑制剂筛选和基因组分析揭示了耐药PDC中的Her2扩增和PI3K突变，以及对KRAS-G12C，PI3K和mTOR三重抑制的脆弱性。此外，我们强调，由Her2扩增驱动的细胞质KRAS定位保持对KRAS-MAPK的低依赖性，从而导致对KRAS抑制剂的内在抗性。

This represents a novel mechanism of resistance. Altogether, the study findings show that KRAS localization may regulate the balance between MAPK and PI3K signaling intensity while providing valuable information for predicting KRAS–MAPK signaling dependency..

这代表了一种新的抵抗机制。总之，研究结果表明，KRAS定位可能调节MAPK和PI3K信号强度之间的平衡，同时为预测KRAS-MAPK信号依赖性提供有价值的信息。。

Methods

方法

Cell lines and culture conditions

KRAS-G12C and -G12D-positive CRC-PDCs and Her2-amplified CRC-PDCs were established from surgically resected tumor specimens. Tumor specimens were provided with informed consent for genetic and cell biological analyses, which were performed following the protocols approved by the Institutional Review Board of the Japanese Foundation for Cancer Research (#2013-1093).

从手术切除的肿瘤标本中建立KRAS-G12C和-G12D阳性CRC pDC和Her2扩增的CRC pDC。肿瘤标本已获得遗传和细胞生物学分析的知情同意，这些分析是按照日本癌症研究基金会机构审查委员会批准的方案进行的（#2013-1093）。

KRAS-G12C-positive-PDCs were cultured in DMEM/F-12, GlutaMAX medium (Thermo Fisher Scientific, Waltham, MA, USA) with 1×STEMPRO hESC SFM (Thermo Fisher Scientific), 1.8% bovine serum albumin (BSA; Thermo Fisher Scientific), 8 ng/ml bFGF (BPS Biosciences, San Diego, CA, USA), 0.1 mM 2-mercaptoethanol (Wako, Osaka, Japan), 10 μM Y-27632 (LC Laboratories, Woburn, MA, USA), and Penicillin-Streptomycin-AmphotericinB Suspension (x 1) (Nacalai Tesque, Kyoto, Japan) (ESC + Y medium).

将KRAS-G12C阳性pDC在DMEM/F-12，GlutaMAX培养基（Thermo Fisher Scientific，Waltham，MA，USA）中与1x STEMPRO hESC SFM（Thermo Fisher Scientific），1.8%牛血清白蛋白（BSA；Thermo Fisher Scientific）一起培养，8ng/ml bFGF（BPS Biosciences，圣地亚哥，加利福尼亚州，美国），，10μMY-27632（LC Laboratories，Woburn，MA，USA）和青霉素-链霉素-两性霉素悬浮液（x 1）（Nacalai Tesque，Kyoto，Japan）（ESC+Y培养基）。

G12D-PDCs were cultured in medium containing equal proportions of Roswell Park Memorial Institute-1640 (RPMI1640) (Wako) and Ham’s F12 (Wako) with 10% FBS, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Nacalai Tesque), and 1× antibiotic-antimycotic mixed stock solution (Wako). HCT116, GP2d, LS180, and AsPC-1 cells purchased from ATCC were cultured in RPMI1640 with 10% FBS and 100 µg/mL of kanamycin (Meiji Seika Pharma, Tokyo, Japan).

G12D pDC在含有等量比例的Roswell Park Memorial Institute-1640（RPMI1640）（Wako）和含10%FBS的Ham's F12（Wako）的培养基中培养，20 mM 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES；Nacalai Tesque）和1x抗生素-抗真菌混合原液（Wako）。将购自ATCC的HCT116，GP2d，LS180和AsPC-1细胞在含有10%FBS和100μg/mL卡那霉素（Meiji Seika Pharma，Tokyo，Japan）的RPMI1640中培养。

H358, SW837, and SW1463 purchased from ATCC were cultured in DMEM low glucose (Wako) supplemented with 10% FBS and 100 µg/mL of kanamycin (Meiji Seika Pharma). All cell lines were authenticated..

将购自ATCC的H358，SW837和SW1463在补充有10%FBS和100µg/mL卡那霉素（Meiji Seika Pharma）的DMEM低葡萄糖（Wako）中培养。所有细胞系均经过鉴定。。

Reagents

试剂

MedChem Express (Monmouth Junction, NJ, USA) and Shanghai Biochempartner (Shanghai, China) supplied sotorasib. While afatinib was purchased from ChemiTek (Esposende, Portugal), cetuximab was purchased from MERCK (Darmstadt, Germany). BEZ235, GDC0941, and PP242 were purchased from Adooq BioScience (Irvine, CA, USA).

MedChem Express（美国新泽西州蒙茅斯交界处）和上海生物化学合作伙伴（中国上海）提供了索托拉西布。阿法替尼购自ChemiTek（葡萄牙埃斯波森德），西妥昔单抗购自默克（德国达姆施塔特）。BEZ235，GDC0941和PP242购自Adooq BioScience（美国加利福尼亚州欧文）。

Supplementary Table .

补充表。

1

1

contains detailed information on the inhibitor library screening drugs.

包含有关抑制剂文库筛选药物的详细信息。

Cell viability assay

细胞活力测定

Commercial cells were seeded in triplicate into 96-well plates (IWAKI, Shizuoka, Japan) at 3000 cells/well for 24 h. PDCs were seeded in triplicate into 96-well collagen-coated plates (IWAKI, Shizuoka, Japan) at 3000 cells/well for 24 h, after which serially diluted inhibitors were cultured in media for an additional 72 h.

将商业细胞一式三份以3000个细胞/孔的速度接种到96孔板（日本静冈岩城）中24 h、 将pDC一式三份以3000个细胞/孔接种到96孔胶原包被的平板（IWAKI，Shizuoka，Japan）中24小时，然后将连续稀释的抑制剂在培养基中再培养72小时。

Cells were then incubated with CellTiter-Glo assay reagent (Promega, Madison, WI, USA), and luminescence was measured using a Tristar LB941 microplate luminometer (Berthold Technologies, La Jolla, CA, USA). To calculate cell viability and the nonlinear regression model with a sigmoidal dose-response curve, we used GraphPad Prism version 8.0 or 9.0 (GraphPad software)..

然后将细胞与CellTiter-Glo测定试剂（Promega，Madison，WI，USA）一起温育，并使用Tristar LB941微孔板发光计（Berthold Technologies，La Jolla，CA，USA）测量发光。为了计算细胞活力和具有S形剂量反应曲线的非线性回归模型，我们使用了GraphPad Prism版本8.0或9.0（GraphPad软件）。。

3D cell viability assay

3D细胞活力测定

3D layered co-culture system was prepared in our previous paper. In brief, 9.0 × 10

在我们之前的论文中制备了3D分层共培养系统。简而言之，9.0×10

5

5

NHDFs and 1.35 × 10

NHDF和1.35×10

4

4

HUVECs were added to a mixture of 150 μL of 0.1 mg/mL heparin/100 mM Tris-HCl (pH 7.4) solution and 150 μL of 0.1 mg/mL collagen/acetic acid (pH 3.7) solution, and centrifuged at 1000 ×g for 2 min at room temperature to obtain viscous material. After suspending the obtained viscous material in DMEM culture medium, the suspension was seeded in 96-well cell culture inserts (#7369, Corning Inc.) and centrifuged at room temperature and 400 ×g for 1 min.

将HUVEC加入150μl0.1mg/mL肝素/100mM Tris-HCl（pH 7.4）溶液和150μl0.1mg/mL胶原/乙酸（pH 3.7）溶液的混合物中，并在室温下以1000×g离心2分钟以获得粘性物质。将获得的粘性物质悬浮在DMEM培养基中后，将悬浮液接种在96孔细胞培养插入物（Corning Inc.）中，并在室温和400×g下离心1分钟。

Then, incubated in a CO2 incubator (37 °C, 5%) for 24 h. Then, 0.5 × 10.

然后，在CO2培养箱（37℃，5%）中孵育24小时。然后，0.5×10。

4

4

or 1.0 × 10

或1.0×10

4

4

cancer cells were suspended in DMEM culture medium and seeded in 96-well cell culture inserts and incubated in a CO2 incubator (37 °C, 5%) for 7 days.

将癌细胞悬浮于DMEM培养基中，接种于96孔细胞培养插入物中，并在CO2培养箱（37℃，5%）中孵育7天。

Then, anticancer drugs or antibodies were added to the cancer co-culture 3D models and incubated for at least 72 h. The effect of the drugs was evaluated by counting the cancer cells as follows. The remaining proliferative cancer cell number was evaluated by measuring a fluorescent intensity by a high-content confocal microscope analysis system (Operetta CLSTM, PerkinElmer Corp.) after immune-fluorescent staining of CK8/18, and Ki67..

然后，将抗癌药物或抗体添加到癌症共培养3D模型中并孵育至少72小时。通过如下计数癌细胞来评估药物的作用。在CK8/18和Ki67的免疫荧光染色后，通过高含量共聚焦显微镜分析系统（Operetta CLSTM，PerkinElmer Corp.）测量荧光强度来评估剩余的增殖性癌细胞数量。。

Immunoblot analysis

免疫印迹分析

Cells were seeded in 6-well or 12-well collagen-coated plates (IWAKI) at 3–5 × 10

将细胞以3-5×10接种在6孔或12孔胶原涂层板（IWAKI）中

5

5

cells/well for 24 h, followed by incubation with each concentration of inhibitors. Cell lysates were then collected at the indicated time points by lysis buffer containing 0.1 M Tris-HCl (pH 7.5), 10% glycerol (Wako), and 1% SDS (Nacalai Tesque) and boiled at 100 °C for 5 min. Protein concentration was determined using a BCA protein assay kit (Thermo Fischer Scientific, Waltham, MA, USA).

细胞/孔24小时，然后与每种浓度的抑制剂孵育。然后通过含有0.1M Tris-HCl（pH 7.5），10%甘油（Wako）和1%SDS（Nacalai Tesque）的裂解缓冲液在指定的时间点收集细胞裂解物，并在100℃煮沸5分钟。使用BCA蛋白质测定试剂盒（Thermo Fischer Scientific，Waltham，MA，USA）测定蛋白质浓度。

Cell lysates were adjusted to 1 µg/µl using lysis buffer, and a 20% volume of sample buffer containing 0.65 M Tris (pH 6.8), 20% 2-mercaptoethanol, 10% glycerol, 3% SDS, and 0.01% bromophenol blue was added. Then, 10 μg of each sample was loaded to perform SDS-polyacrylamide gel electrophoresis and immunoblotting using following antibodies..

使用裂解缓冲液将细胞裂解物调节至1μg/μl，并使用20%体积的含有0.65的样品缓冲液 加入M Tris（pH 6.8），20%2-巯基乙醇，10%甘油，3%SDS和0.01%溴酚蓝。然后，加载每个样品10μg，使用以下抗体进行SDS-聚丙烯酰胺凝胶电泳和免疫印迹。。

Total S6 ribosomal protein (#2217, 1:2000), phospho-S6 ribosomal protein (#5364, 1:2000), total p42/44 ERK/MAPK (#9102, 1:2000), phospho-p42/44 ERK/MAPK (#9101, 1:2000), total AKT (#4691, 1:1000), phospho-AKT (T308) (#2965, 1:1000), phospho-AKT (S473) (#4060, 1:1000), total EGFR (#2646, 1:1000), Her2 (#2165, 1:1000), phospho-Her2 (#2243, 1:1000), Her3 (#12708, 1:1000), phosphor-Her3 (#2842, 1:1000), PI3 Kinase p110α (#4249, 1:1000), poly (ADP-ribose) polymerase (PARP) (#9542, 1:1000), cleaved PARP (#9541, 1:1000), Cas9 (#14697, 1:1000) were purchased from Cell Signaling Technology.

总S6核糖体蛋白（#2217，1:2000），磷酸S6核糖体蛋白（෬5364，1:2000），总p42/44 ERK/MAPK（෬9102，1:2000），磷酸p42/44 ERK/MAPK（෬9101，1:2000），总AKT（෬4691，1:1000），磷酸AKT（T308）（෬2965，1:1000），磷酸AKT（S473）（෬4060，1:1000），总EGFR（෬4691，1:1000）#2646，1:1000），Her2（#2165，1:1000），磷酸化Her2（#2243，1:1000），Her3（#12708，1:1000），磷酸化Her3（#2842，1:1000），PI3激酶p110α（#4249，1:1000），聚（ADP-核糖）聚合酶（PARP）（#9542，1:1000），裂解的PARP（3541，1:1000），Cas9（#14697，1000）1:1000）购自Cell Signaling Technology。

phospho-EGFR (Y1068) antibody (GTX132810, 1:1000) was purchased from Gene Tex (Irvine, CA, USA), KRAS antibody (WH0003845M1, 1:1000) was purchased from Sigma (SIGMA ALDRICH, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (MAB374, 1:10000) was purchased from Millipore (Burlington, MA, USA).

磷酸-EGFR（Y1068）抗体（GTX132810，1:1000）购自Gene Tex（Irvine，CA，USA），KRAS抗体（WH0003845M1，1:1000）购自Sigma（Sigma-ALDRICH，St.Louis，MO，USA）和甘油醛3-磷酸脱氢酶（GAPDH）抗体（MAB374，1:10000）购自Millipore（Burlington，MA，USA）。

For signal detection, ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fischer Scientific) were used. Amersham Imager 600 (GE Healthcare) or Amersham Imager 800 (GE Healthcare) was used to detect chemiluminescent signals..

对于信号检测，使用ECL Prime Western印迹检测试剂（GE Healthcare，Chicago，IL，USA）和SuperSignal West Femto最大灵敏度底物（Thermo Fischer Scientific）。Amersham Imager 600（GE Healthcare）或Amersham Imager 800（GE Healthcare）用于检测化学发光信号。。

Ras pull-down assay

Ras下拉测定

In total 3–5 × 10

总共3-5个带宽10

6

6

cells were seeded in 10 cm collagen-coated dish for 24 h. Sotorasib or MRTX1133 was then treated for an indicated time point. Total proteins were collected using a Ras-Activating Assay Kit (Millipore), and GTP-Ras detection was conducted as follows: Briefly, cell lysates were collected with lysis buffer, protein concentration was measured by BCA protein assay kit, and 500 µg of the protein lysates were incubated with Raf1-RBD agarose beads for 1.

将细胞接种在10cm胶原包被的培养皿中24小时。然后将Sotorasib或MRTX1133处理指定的时间点。使用Ras激活测定试剂盒（Millipore）收集总蛋白质，并如下进行GTP-Ras检测：简言之，用裂解缓冲液收集细胞裂解物，通过BCA蛋白质测定试剂盒测量蛋白质浓度，并将500μg蛋白质裂解物与Raf1 RBD琼脂糖珠孵育1。

The beads were washed with lysis buffer and resuspended SDS sample buffer, after which GTP-Ras proteins were collected by boiling..

用裂解缓冲液和重悬SDS样品缓冲液洗涤珠子，然后通过煮沸收集GTP-Ras蛋白。。

siRNA knockdown

siRNA敲低

Cells were transfected with 20 nM of each siRNA using Lipofectamin RNAiMAX Transfection Reagent (Thermo Fisher Scientific) and OPTI-MEM (Thermo Fisher Scientific). For the cell viability assay, cells were seeded in triplicate into 96-well collagen-coated plates at 3000 cells/well and incubated with siRNA mixture for 72 h.

使用Lipofectamin RNAiMAX转染试剂（Thermo Fisher Scientific）和OPTI-MEM（Thermo Fisher Scientific），用20nm的每种siRNA转染细胞。对于细胞活力测定，将细胞一式三份以3000个细胞/孔接种到96孔胶原包被的平板中，并与siRNA混合物孵育72小时。

Cell viability was measured using the CellTiter-Glo assay reagent (Promega). For immunoblot analysis, cells were seeded at 5 × 10.

使用CellTiter-Glo测定试剂（Promega）测量细胞活力。为了进行免疫印迹分析，将细胞接种在5×10。

5

5

cells/well in 6-well collagen-coated and incubated with siRNA mixture for 48 h. Cell lysate was collected and analyzed by immunoblotting. The following siRNAs were purchased from Dharmacon (Lafayette, CO, USA).

细胞/孔在6孔胶原包被并与siRNA混合物孵育48小时。收集细胞裂解物并通过免疫印迹分析。以下siRNA购自Dharmacon（Lafayette，CO，USA）。

ON-TARGET plus Non-targeting Pool (D-001810-10-05)

目标加非目标池（D-001810-10-05）

UGGUUUACAUGUCGACUAA,

ugguuaacaguggacua，

UGGUUUACAUGUUGUGUGA,

你的小，

UGGUUUACAUGUUUUCUGA,

你的生活，

UGGUUUACAUGUUUUCCUA

危险

ON-TARGET plus Human KRAS (3845) siRNA (LQ-005069-00-0005)

si-KRAS#1: GGAGGGUUUCUUUGUGUA

CRAS#1：GUG

si-KRAS pool: GAAGUUAUGGAAUUCCUUU, GAGAUAACACGAUGCGUAU

KRAS游泳池：高加索乌，高加索乌

ON-TARGET plus Human PIK3CA (5290) siRNA (LQ-003018-00-0005)

靶向加人PIK3CA（5290）siRNA（LQ-003018-00-0005）

si-PIK3CA#1: GCGAAAUUCUCACACUAUU

和-PIK3CA#1:GCGAAAUUCACACUAUU

si-PIK3CA#4: GACCCUAGCCUUAGAUAAA

si-PIK3CA#4:GACCCUAGCCUUAGAAA

Inhibitor library screening

抑制剂文库筛选

Cells were seeded in duplicate into 96-well collagen-coated plates at 3000 cells/well for 24 h, after which indicated inhibitors with KRAS inhibitors were co-cultured for an additional 72 h. Subsequently, cell viability was measured using the CellTiter-Glo assay reagent. Relative cell viability to a single treatment of KRAS inhibitors was calculated..

将细胞一式两份以3000个细胞/孔接种到96孔胶原包被的平板中24小时，然后将指示的抑制剂与KRAS抑制剂共培养另外72小时。随后，使用CellTiter-Glo测定试剂测量细胞活力。计算了单次治疗KRAS抑制剂的相对细胞活力。。

Immunofluorescence staining

免疫荧光染色

Cells were cultured in a Lab-tec chamber slide (Thermo Fisher Scientific) for 24 h and fixed with 4% paraformaldehyde (Wako) for 20 min at room temperature. Cells were then permeabilized for 1 h by blocking and permeabilizing buffer containing Blocking One (Nacarai Tesque) and 0.1% Triton X-100 (Sigma) with PBS (Wako).

将细胞在Lab-tec室载玻片（Thermo Fisher Scientific）中培养24小时，并在室温下用4%多聚甲醛（Wako）固定20分钟。然后通过用PBS（Wako）封闭和透化含有封闭一（Nacarai Tesque）和0.1%Triton X-100（Sigma）的缓冲液使细胞透化1小时。

Subsequently, the cells were incubated with an antibody solution containing primary antibodies with 0.1% BSA (Thermo Fisher Scientific), 0.3% Triton-X-100 (Sigma), and PBS (Wako) at 4°C overnight. Cells were washed with PBS three times and incubated with secondary antibody and antibody solution at room temperature for 1 h, followed by Hoechst staining for 10 min.

随后，将细胞与含有0.1%BSA（Thermo Fisher Scientific），0.3%Triton-X-100（Sigma）和PBS（Wako）的一抗的抗体溶液在4°C孵育过夜。将细胞用PBS洗涤三次，并与二抗和抗体溶液在室温下孵育1小时，然后进行Hoechst染色10分钟。

The chamber slide was washed and mounted by mounting media (Thermo Fisher Scientific). Images were captured with confocal microscopy LSM880 with 60x or 100x objective (Zeiss) and analyzed using ImageJ. The following primary and secondary antibodies were used in immunofluorescence staining: Anti-KRAS (WH0003845M1, SIGMA, 1:1000), E-cadherin (#3195, CST, 1:1000), Her2 (ab214275, Abcam, 1:1000), Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A11008, Thermo Fisher Scientific, 1:1000), and Alexa Fluor 647 goat anti-mouse IgG (H + L) (A21236, Thermo Fisher Scientific, 1:1000); Hoechst 33342 (H1399, Thermo Fisher Scientific, 1:5000)..

清洗腔室载玻片并通过安装介质（Thermo Fisher Scientific）安装。使用具有60倍或100倍物镜（Zeiss）的共聚焦显微镜LSM880捕获图像，并使用ImageJ进行分析。以下一抗和二抗用于免疫荧光染色：抗KRAS（WH0003845M1，SIGMA，1:1000），E-钙粘蛋白（3195，CST，1:1000），Her2（ab214275，Abcam，1:1000），Alexa Fluor 488山羊抗兔IgG（H + L）（A11008，Thermo Fisher Scientific，1:1000）和Alexa Fluor 647山羊抗小鼠IgG（H + L）（A21236，Thermo Fisher Scientific，1:1000）；Hoechst 33342（H1399，Thermo Fisher Scientific，1:5000）。。

Establishment of CRISPR-Cas9-mediated Her2 knockout cells

CRISPR-Cas9介导的Her2基因敲除细胞的建立

The sgRNA sequence for ERBB2 was designed using CRISPR Knockout Pooled Library (GeCKO v2) and target sequence cloned into LentiCRISPRv2, which was received from Addgene (#52961). Lentivirus mixtures were produced by ViraPower (Thermo Fisher Scientific) using 293FT cells. Cells were infected using a lentivirus-containing medium supplemented with polybrene (8 mg/ml).

使用CRISPR敲除合并文库（GeCKO v2）设计ERBB2的sgRNA序列，并将靶序列克隆到LentiCRISPRv2中，该序列是从Addgene（#52961）获得的。ViraPower（Thermo Fisher Scientific）使用293FT细胞生产慢病毒混合物。使用补充有聚凝胺（8mg/ml）的含慢病毒的培养基感染细胞。

After 24 h, the infected cells were selected by puromycin (10 µg/ml)..

24小时后，通过嘌呤霉素（10µg/ml）选择感染的细胞。。

Target sequencing analysis

目标测序分析

Genomic DNA was obtained from surgically resected tumor specimens using DNeasy® Blood&Tissue kit (QIAGEN, Hilden, Germany). For cancer-related gene-focused sequencing, a Haloplex custom panel was used, and the details are listed in Supplementary Table

使用DNeasy®血液和组织试剂盒（QIAGEN，Hilden，Germany）从手术切除的肿瘤标本中获得基因组DNA。对于与癌症相关的基因聚焦测序，使用了Haloplex定制面板，详细信息列于补充表中

2

2

(illumine, San Diego, CA, USA). Paired-end sequencing was executed in the NovaSeq6000 platform. Illumina adapter sequences and low-quality bases were trimmed using Trimmomatic-0.39 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:30 MINLEN:40

（illumine，美国加利福尼亚州圣地亚哥）。配对末端测序是在NovaSeq6000平台上执行的。使用Trimmomatic-0.39修剪Illumina适配器序列和低质量碱基，前导：20尾随：20滑动窗口：4:30分钟：40

49

49

. Then, passed-reads were mapped onto the human genome (GRCh38/hg38) using HISAT2 (Version 2.1.0) and a BAM file was obtained using SAMtools (Version 1.8)

然后，使用HISAT2（版本2.1.0）将通过的读物映射到人类基因组（GRCh38/hg38）上，并使用SAMtools（版本1.8）获得BAM文件

50

50

,

,

51

51

. More than 1% of SNPs and indels were detected from GATK (Version 4.1.8.0) and VarScan (Version 2.4.4)

。从GATK（版本4.1.8.0）和VarScan（版本2.4.4）中检测到超过1%的SNP和插入缺失

52

52

,

,

53

53

. A graphical image of gene mutations was obtained from maftools

.基因突变的图形图像是从maftools获得的

54

54

. The BAM file was also used for copy number calling, and a graphical image of Chr17 was obtained from CNVkit (Version 0.9.8).

BAM文件也用于拷贝数调用，并且从CNVkit（版本0.9.8）获得了Chr17的图形图像。

Exome sequencing analysis

外显子组测序分析

Genomic DNA was obtained from surgically resected tumor specimens using DNeasy® Blood&Tissue kit (QIAGEN). Library preparation was performed using SureSelect Human V6 (illumine, San Diego, CA, USA), and paired-end sequencing NGS was executed in the NovaSeq6000 platform. Illumina adapter sequences and low-quality bases were trimmed using Trimmomatic-0.39 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:30 MINLEN:40.

使用DNeasy®血液和组织试剂盒（QIAGEN）从手术切除的肿瘤标本中获得基因组DNA。使用SureSelect Human V6（illumine，圣地亚哥，加利福尼亚，美国）进行文库制备，并在NovaSeq6000平台上执行配对末端测序NGS。使用Trimmomatic-0.39修剪Illumina适配器序列和低质量碱基，前导：20尾随：20滑动窗口：4:30分钟：40。

49

49

. Then, passed-reads were mapped onto the human genome (GRCh38/hg38) using HISAT2 (Version 2.1.0) and a BAM file was obtained using SAMtools (Version 1.8)

然后，使用HISAT2（版本2.1.0）将通过的读物映射到人类基因组（GRCh38/hg38）上，并使用SAMtools（版本1.8）获得BAM文件

50

50

,

,

51

51

. The BAM file was used for copy number calling, and a graphical image of Chr17 was obtained from CNVkit (Version 0.9.8).

。BAM文件用于拷贝数调用，并且从CNVkit（版本0.9.8）获得了Chr17的图形图像。

In vivo study

All in vivo studies were approved by the Institutional Animal Care and Use Committee approved and conducted according to the institutional guidelines. 5–10 × 10

所有体内研究均经机构动物护理和使用委员会批准，并根据机构指南进行。5-10××10

6

6

cells for JC288 and JC261 cells were subcutaneously injected into BALB/c-nu/nu mice (Charles River Laboratories, Wilmington, MA, USA). After the tumor volumes reached 100–200 mm3, the mice were orally administered (sotorasib) or intraperitoneally administered (cetuximab) for the indicated days. The drugs were dissolved with the following solution: 2 w/v % HPMC (SIGMA ALDRICH) and 1% Tween80 (Nacalai Tesque).

将JC288和JC261细胞的细胞皮下注射到BALB/c-nu/nu小鼠（Charles River Laboratories，Wilmington，MA，USA）中。肿瘤体积达到100-200mm3后，将小鼠口服（索托拉西布）或腹膜内（西妥昔单抗）指定天数。用以下溶液溶解药物：2 w/v%HPMC（SIGMA-ALDRICH）和1%吐温80（Nacalai Tesque）。

Tumor size and body weight were measured more than twice a week. The tumor volume was calculated as 0.5 × length × width.

每周测量两次以上的肿瘤大小和体重。肿瘤体积计算为0.5×长度×宽度。

2

2

. The mice were sacrificed when tumor volume reached humane endpoint (1000 mm

当肿瘤体积达到人类终点（1000毫米）时，处死小鼠

3

3

) by cervical dislocation. Significant differences in tumor volume were calculated using the Mann–Whitney

)颈椎脱位。使用Mann-Whitney计算肿瘤体积的显着差异

U

U

test with GraphPad Prism version 9.0.

使用GraphPad Prism 9.0版进行测试。

Data availability

数据可用性

We have deposited the original fastq files in this article to the DNA DataBank of Japan (NBDJ) ; JGAS000767. The original fastq files within the article are available upon reasonable request. All the somatic mutation data from target re-sequence was attached as the Supplementary data.

我们已将本文中的原始fastq文件保存到日本DNA数据库（NBDJ）；JGAS000767。本文中的原始fastq文件可根据合理要求提供。。

Code availability

代码可用性

No code or scripts are used in this study.

本研究未使用任何代码或脚本。

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Acknowledgements

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We thank Dr. Ai Takemoto (JFCR) for the in vivo study and Ms. Akiko Uotu, Ms. Ramu Inoue, and Ms. Mai Suzuki for the preparation of patient derived cells, and Dr. Yosuke Seto for helping NGS analysis. This study was supported in part by MEXT/JSPS KAKENHI grant number JP23KJ0562 (to K.M.), and JP22K18383 and JP24K02333 (to R.K.), and the grant from the AMED grant number JP24am0521012s0101, JP24ama221231h0001, JP24ama221210h0003 and JP24ck0106795h0002 (to R.K.) and the grants from the Naito Foundation (to R.K.), Chugai Foundation for Innovative Drug Discovery Science (to R.K.), Mitsubishi Foundation (to R.K), and the grant from Nippon Foundation and Takeda Science Foundation..

我们感谢Ai Takemoto博士（JFCR）的体内研究，感谢Akiko Uotu女士，Ramu Inoue女士和Mai Suzuki女士制备患者来源的细胞，感谢Yosuke Seto博士帮助NGS分析。这项研究得到了MEXT/JSPS KAKENHI资助号JP23KJ0562（授予K.M.）、JP22K18383和JP24K02333（授予R.K.）的部分支持，以及AMED资助号JP24am0521012s0101、JP24ama221231h0001、JP24AMA22110H0003和JP24ck0106795h0002（授予R.K.）的资助，以及奈藤基金会（授予R.K.）、中盖创新药物发现科学基金会（授予R.K.）、三菱基金会（授予R.K）的资助，以及日本基金会和武田科学的资助基金会。。

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Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan

日本东京日本癌症研究基金会癌症化疗中心实验化疗部

Kohei Maruyama, Yuki Shimizu, Tomoko Oh-hara & Ryohei Katayama

丸山浩平，[UNK]YukiShimizu，[UNK]友子Oh hara[UNK]&[UNK].片山良平

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan

东京大学前沿科学研究生院计算生物学与医学系，日本东京

Kohei Maruyama & Ryohei Katayama

丸山光平&片山良平

Business Development Division, Technical Research Institute, TOPPAN Holdings Inc., Saitama, Japan

日本埼玉县TOPPAN Holdings Inc.技术研究所业务发展部

Yumi Nomura & Yuki Takahashi

野村由美和高桥由纪

Division of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan

日本东京日本癌症研究基金会癌症化疗中心临床化疗部

Yumi Nomura & Yuki Takahashi

野村由美和高桥由纪

Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan

日本京都京都大学医学研究生院外科

Satoshi Nagayama

长山聪

Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan

日本东京日本癌症研究基金会癌症研究所医院胃肠外科

Satoshi Nagayama

长山聪

Department of Surgery, Uji-Tokushukai Medical Center, Kyoto, Japan

Satoshi Nagayama

长山聪

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan

日本东京日本癌症研究基金会癌症化疗中心

Naoya Fujita

藤田直也

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Kohei Maruyama

丸山光平

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Yuki Shimizu

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Contributions

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K.M., Y.S., S.N., N.F., and R.K. designed the study. K.M., Y.S., Y.N., T.O., Y.T., and R.K. conducted the experiments and collected data. K.M., Y.S., Y.N., T.O., Y.T., S.N. and R.K. analyzed the data. S.N. served as surgery and take informed consent from patients, and collected colorectal surgical specimens.

K、 M.，Y.S.，S.N.，N.F。和R.K.设计了这项研究。K、 M.，Y.S.，Y.N.，T.O.，Y.T。和R.K.进行了实验并收集了数据。K、 M.，Y.S.，Y.N.，T.O.，Y.T.，S.N.和R.K.分析了数据。S、 N.担任手术并征得患者的知情同意，并收集结直肠手术标本。

K.M., Y.T., S.N. and R.K. wrote the manuscript. All the authors have provided a critical review of the manuscript. K.M., N.S., N.F., and R.K. prepared research grants for this study..

K、 M.，Y.T.，S.N.和R.K.撰写了手稿。所有作者都对手稿进行了批判性审查。K、 M.，N.S.，N.F。和R.K.为这项研究准备了研究资助。。

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Ryohei Katayama

片山良平

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Competing interests

相互竞争的利益

R. Katayama is an Associate Editor for npj Precision Oncology. R. Katayama received research grants from Chugai, TOPPAN Inc. N. Fujita received research grants from TOPPAN Inc. Yuki Takahashi and Yumi Nomura belong to TOPPAN Inc. All other authors declare no conflict of interest.

R、 Katayama是npj Precision Oncology的副主编。R、 Katayama获得了Chugai，TOPPAN Inc.的研究资助。N.Fujita获得了TOPPAN Inc.的研究资助。高桥由纪和野村由美属于TOPPAN Inc.所有其他作者均声明没有利益冲突。

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Supplementary information

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Supplementary Figures

补充数字

Maruyama K et al Supplemental Table 1

Maruyama K等人补充表1

Maruyama K et al Supplemental Table 2

Maruyama K等人补充表2

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引用本文

Maruyama, K., Shimizu, Y., Nomura, Y.

丸山，K.，清水，Y.，野村，Y。

et al.

等人。

Mechanisms of KRAS inhibitor resistance in KRAS-mutant colorectal cancer harboring Her2 amplification and aberrant KRAS localization.

携带Her2扩增和异常KRAS定位的KRAS突变结直肠癌中KRAS抑制剂耐药的机制。

npj Precis. Onc.

不，没错。Onc。

9

9

, 4 (2025). https://doi.org/10.1038/s41698-024-00793-6

, 4 (2025).https://doi.org/10.1038/s41698-024-00793-6

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Received

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30 October 2023

2023年10月30日

Accepted

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19 December 2024

2024年12月19日

Published

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:

06 January 2025

2025年1月6日

DOI

DOI

:

:

https://doi.org/10.1038/s41698-024-00793-6

https://doi.org/10.1038/s41698-024-00793-6

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