一、简介

1. Introduction

In humans, patients with pancreatic ductal adenocarcinomas (PDACs) commonly have a poor prognosis. As reported in 2018, the five-year survival rate of PDAC patients is only 9% [1]. The biology of PDAC is aggressive, and a certain portion of patients will die from disease-related complications rather than this disease itself [2]. Traditional approaches for managing this cancer include surgery, radiotherapy and chemotherapy. To exploit the genomic characteristics of PDAC, some molecular targeted approaches have been developed. These approaches have exhibited therapeutic effects in a small portion of metastatic cases carrying specific driver alterations, such as the treatment of cases with a germline BRCA1 or BRCA2 mutation using olaparib, a PARP inhibitor, or the treatment of cases with NTRK gene fusions using larotrectinib or entrectinib [3]. Recently, immune checkpoint blockade (ICB) therapy has opened a new era in the comprehensive treatment of cancers. In metastatic PDAC, only patients with the high microsatellite instability (MSI-H) or deficient mismatch repair (dMMR) phenotype in their tumors are reported to benefit from the ICB therapy with pembrolizumab, an anti-PD-1 drug [4]. However, the MSI-H/dMMR phenotype is rarely detected in PDAC. For those patients without the MSI-H/dMMR phenotype, available data indicate that their responses to monotherapy by using ICB drugs are extremely poor [5].

The existing immune environment in tumors will impact the effectiveness of ICB therapy [6]. In PDAC, the tumor milieu is generally immunosuppressive [7]. Recently, driver oncogenes have been recognized to play a convincing role in the cancer immune status [8]. In PDAC, the Kirsten rat sarcoma virus oncogene homolog (KRAS) gene is broadly mutated [9]. KRAS mutations in PDAC include those induced by a missense mutation in codon 12 or codon 13, leading to a replacement of the original glycine (G) by other amino acids, thus causing persistent activation of the KRAS protein in this setting [9]. The KRAS mutation acts as a driver to cause PDAC occurrence and progression together with the concomitant inactivation of other genes, such as TP53, CDKN2A and SMAD4 [10, 11] (Figure 1). In this process, the KRAS mutation will also lead to activation of downstream pathways that can improve cancer cell survival, proliferation, immune evasion and drug resistance [7, 9]. Concerning immunosuppression in PDAC, the KRAS mutation utilizes several routes to achieve this goal, such as activating the yes-associated protein (YAP)-TAZ pathway and its downstream JAK-STAT3 signaling [12], inducing cell autophagy-associated MHC-I degradation by reprogramming glucose metabolism [13, 14], and synergizing with other genetic alterations (e.g., TP53 inactivation) [15] (Figure 1). Consequently, PDAC tumors can be infiltrated by myeloid cells with pro-cancer functions, such as neutrophils, myeloid-derived suppressive cells (MDSCs) and M2-like macrophages [7].

Figure 1. The note chart of KRAS mutation-induced growth and immunosuppression in PDAC tumors. The KRAS mutation causes a suppressive milieu in PDAC tumors mainly via the following routes, such as activation of MAPK and PI3K-Akt, activation of YAP-TAZ and JAK-STAT3, and induction of cell autophagy and metabolic reprogramming in PDAC cells. In this context, the survival and proliferation of PDAC cells will be accelerated, and an overgrowth of tumor cells can cause a hypoxia within the tumor, which then activates HIF-1α to upregulate the expression of gene encoding VEGF by PDAC cells. VEGF is a potent cytokine that induces angiogenesis and immune evasion (e.g. PD-L1 upregulation and tumoricidal T cell exhaustion). Meanwhile, PDAC cells can increase their production of suppressive cytokines and chemokines, such as IL-4, IL-6, IL-13, CSF-1 and MCP-1, which then recruit and increase the survival and suppressive function of immune infiltrates including CAFs, MDSCs, M2-like TAMs, IDO-producing DCs and Treg cells. In this context, an overload of suppressive cells will increase the local levels of suppressive cytokines and chemokines, such as TGF-β, IDO, IL-10, GM-CSF, CXCL1, CXCL8, CXCL12 and CXCL13, thus strengthening the immunosuppression in the tumor (e.g. tumoricidal T cell exclusion). In concert with the KRAS mutation, other alterations at genetic and molecular levels, such as LKB1 inactivation, TP53 inactivation, PTEN inactivation, FAK activation, PIK3CA activation or WNT activation, also contribute to the tumor growth (e.g. PDAC cell survival, proliferation and invasion) and immune evasion (PD-L1 upregulation). .

In addition to PDAC, other cancers in humans, such as colorectal adenocarcinomas (CRACs) and lung adenocarcinomas (LUACs), also harbor a high prevalence of KRAS mutations [9]. Although KRAS mutation has been revealed to correlate with immune evasion in PDAC [13, 14], the situation in LUAC appears to be different because LUAC tumors with KRAS mutation plus TP53 inactivation commonly have massive infiltration of tumoricidal T cells and PD-L1 upregulation [16]. Moreover, clinical data support that LUAC patients with this pattern of tumor immunity can largely benefit from anti-PD-1 monotherapy [17]. Similarly, KRAS mutation is able to cause immunosuppression in CRAC tumors as well. However, unlike in PDAC, the published data suggest that CRAC patients with this phenotype can benefit from a combinational strategy featuring conventional therapy plus an ICB drug [18]. Importantly, despite having KRAS mutation, PDACs, CRACs and LUACs differ in their tumor immune status (Table 1).

在人类中，胰腺导管腺癌（PDAC）患者通常预后较差。肿瘤中现有的免疫环境会影响ICB治疗的有效性[ 6 ]。在 PDAC 中，肿瘤环境通常具有免疫抑制作用 [ 7 ]。最近，人们已经认识到驱动癌基因在癌症免疫状态中发挥着令人信服的作用 [ 8 ]。在 PDAC 中，Kirsten 大鼠肉瘤病毒癌基因同源物 ( KRAS ) 基因发生广泛突变 [ 9 ]。PDAC 中的KRAS突变包括由密码子 12 或密码子 13 中的错义突变引起的突变，导致原始甘氨酸 (G) 被其他氨基酸取代，从而导致持续激活在这种情况下的KRAS蛋白 [ 9 ]。的KRAS突变作为驾驶员原因PDAC发生，发展与其它基因，如肿瘤蛋白P53基因（的伴随灭活TP53），细胞周期蛋白依赖性激酶抑制剂2A基因（CDKN2A）和SMAD家族成员4基因（SMAD4） [ 10 , 11 ]（图 1）。在这个过程中，KRAS突变也会导致下游通路的激活，这些通路可以提高癌细胞的存活、增殖、免疫逃避和耐药性[ 7 , 9 ]。关于 PDAC 中的免疫抑制，KRAS突变利用多种途径来实现这一目标，例如激活 yes 相关蛋白 (YAP)-tafazzin (TAZ) 通路及其下游 Janus 激酶信号转导和转录激活因子 3 (JAK-STAT3) 信号传导 [ 12 ]，诱导细胞通过重新编程的葡萄糖代谢[自噬相关的主要组织相容性复合体I（MHC-I）降解13，14 ]，并与其他的遗传改变协同（例如，TP53失活）[ 15 ]（图1）。因此，PDAC 肿瘤可以被具有促癌功能的骨髓细胞浸润，例如中性粒细胞、骨髓源性抑制细胞 (MDSCs) 和 M2 样巨噬细胞 [ 7]]。Table 1. The comparison of immune-related characteristics among KRAS-mutant adenocarcinomas

图 1. PDAC 肿瘤中KRAS突变诱导生长和免疫抑制的注释图。该KRAS突变主要通过以下途径在 PDAC 肿瘤中引起抑制环境，例如激活丝裂原活化蛋白激酶 (MAPK) 和磷脂酰肌醇 3-激酶 (PI3K)-Akt，激活 YAP-TAZ 和 JAK-STAT3，以及诱导PDAC细胞中的细胞自噬和代谢重编程。在这种情况下，PDAC细胞的存活和增殖会加速，肿瘤细胞的过度生长会导致肿瘤内缺氧，进而激活缺氧诱导因子1（HIF-1）α，上调编码基因的表达。血管内皮生长因子 (VEGF) 通过 PDAC 细胞。VEGF 是一种有效的细胞因子，可诱导血管生成和免疫逃避（例如，程序性死亡配体 1 (PD-L1) 上调和杀伤肿瘤性 T 细胞耗竭）。同时，PDAC细胞可以增加抑制性细胞因子和趋化因子的产生，例如白介素4（IL-4），IL-6，IL-13，巨噬细胞集落刺激因子1（CSF-1）和单核细胞趋化蛋白1（MCP-1） )，然后招募并增加免疫浸润物的存活和抑制功能，包括癌症相关成纤维细胞 (CAF)、MDSC、M2 样肿瘤相关巨噬细胞 (TAM)、产生吲哚胺 2, 3-双加氧酶 (IDO) 的树突细胞(DC) 和调节性 T 细胞 (Treg 细胞)。在这种情况下，抑制性细胞的过载将增加抑制性细胞因子和趋化因子的局部水平，例如转化生长因子-β (TGF-β)、IDO、IL-10、粒细胞-巨噬细胞集落刺激因子 (GM-CSF) , 趋化因子 CXC 基序配体 1 (CXCL1)、CXCL8、CXCL12 和 CXCL13，从而加强肿瘤中的免疫抑制（例如，杀瘤性 T 细胞排除）。与KRAS突变，基因和分子水平的其他改变，例如肝激酶 B1 基因 ( LKB1 ) 失活、TP53失活、磷酸酶和张力蛋白同源基因 ( PTEN ) 失活、粘着斑激酶 (FAK) 激活、磷脂酰肌醇 4, 5-二磷酸3-激酶催化亚基 α (PIK3CA) 激活或 Wingless/整合 (WNT) 激活也有助于肿瘤生长（例如 PDAC 细胞存活、增殖和侵袭）和免疫逃避（PD-L1 上调）。PDAC: pancreatic ductal adenocarcinoma; CRAC: colorectal adenocarcinoma; EMT: epithelial-mesenchymal transition; LUAC: lung adenocarcinoma; pMMR: proficient mismatch repair; MAPK: mitogen-activated protein kinase; MSS: microsatellite stability; dMMR: deficient mismatch repair; MSI-H: high microsatellite instability; ICB: immune checkpoint blockade; NM: no mention; PD-L1: programmed death-ligand 1; TP53: tumor protein P53 gene; LKB1: liver kinase B1 gene.

2. KRAS突变在 PDAC 中的致癌作用

Given the above information, this review will focus on the role of KRAS mutation in dictating pancreatic carcinogenesis and the cancer immune status in PDAC, aiming to illustrate the response of PDAC to ICB therapy in published data and to provide new insights into the use of ICB therapy in PDAC treatment. In addition, we will consider other KRAS-mutant cancers, such as CRAC and LUAC, and compare them with PDAC, aiming to uncover the mechanism by which KRAS mutation dictates the cancer immune status across these adenocarcinomas.

人PDAC只具有KRAS突变而不是神经母细胞瘤RAS病毒癌基因同系物基因（NRAS）或哈维大鼠肉瘤病毒原癌基因同源基因（HRAS）突变[ 9 ]。总体而言，97.7% 的 PDAC 病例被检测到具有KRAS突变 ^{[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94][95]}。G12D、G12V和G12R是PDAC中三种最常见的KRAS突变错义形式，其中以G12D错义突变最为常见[ 9 ]（表1）。生理上，正常的KRAS蛋白具有 GTPase 活性，但这些错义变体会产生与 GTP 稳定结合的 KRAS 蛋白，从而组成性激活 MAPK 和 PI3K-Akt 通路，这两条经典通路负责维持细胞存活和增殖 [ 35 ]（图 1）。

2. The carcinogenic role of KRAS mutation in PDAC

Human PDAC exclusively has KRAS mutation rather than NRAS or HRAS mutation [9]. Overall, 97.7% of PDAC cases are detected to have the KRAS mutation [9]. G12D, G12V and G12R are the three most common missense forms of KRAS mutation in PDAC, while the G12D missense mutation is the most frequent among them [9] (Table 1). Physiologically, the normal KRAS protein has GTPase activity, but these missense variants generate a KRAS protein that stably binds with GTP, thus constitutively activating MAPK and PI3K-Akt pathways, two classical pathways responsible for maintaining cell survival and proliferation [19] (Figure 1). For example, in mice bearing PDAC, the KRAS^G12D mutation was revealed to activate the MAPK and PI3K-Akt pathways to increase the cellular content of Krüppel-like factor 5 (KLF5), which was required for PDAC cell proliferation [20]. Consistently, in human cell lines, MAPK activation upon KRAS mutation was revealed to induce posttranscriptional modification of YAP, and KRAS mutation was able to augment the transcriptional activity of YAP on its target genes [21]. Functionally, YAP-TAZ activation was demonstrated to be required for pancreatic carcinogenesis in mice carrying the KRAS^G12D mutation: YAP and TAZ protein levels were upregulated in each stage of PDAC pathogenesis, including pancreatitis, acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), and double knockout of Yap and Taz genes significantly mitigated KRAS^G12D mutation-induced ADM and PanIN lesions [12]. In fact, YAP is essential for maintaining glucose metabolism in normal pancreatic epithelial cells [21]. This means that the KRAS mutation potentially induces a metabolic dysregulation of glucose. In a previous study, the KRAS^G12D mutation was revealed to induce an upregulation of the gene encoding NIX, a critical protein for inducing mitophagy, thus restricting glucose flux into mitochondria (Figure 1). Via this mechanism, glucose metabolism in PDAC cells could be switched to favor glycolysis, and the antioxidant program could be activated, thus facilitating cell proliferation [13]. To understand the relationship among KRAS mutation, the antioxidant program and cell proliferation in PDAC, another study conducted by the same team reported that the KRAS^G12D mutation could activate the Nrf2-related antioxidant program in pancreatic epithelial cells of mice; in addition, PanIN cells from Nrf2-deficient mice were less proliferative than those without Nrf2 deficiency [22]. Consistent with this finding, inhibiting glutathione synthesis in PanIN cells without Nrf2 deficiency decreased their proliferation [22]. Collectively, these results show that KRAS mutation impacts the proliferation of PDAC cells in a metabolic manner (Figure 1).

3. The KRAS mutation and immune environment in PDAC

This result suggests that Yap is required for KRAS mutation-induced immunosuppression in PDAC tumors.

4. Current status of immune checkpoint blockade therapy for PDAC

The effectiveness of ICB therapy on PDAC

Abbreviation: PDAC: pancreatic ductal adenocarcinoma; ORR: objective response rate; SBRT:

: The total ORR of four arms;

: 67% of them received at least one line of chemotherapy; ^C: The total ORR of three arms.

In fact, metastatic cancers commonly show clonal evolution of tumor cells as the therapies are engaged [49], and this scenario is suitable for ICB therapy [50]. As reported, first-line chemotherapy using [FOLFIRINOX] (oxaliplatin plus irinotecan plus 5-fluorouracil plus calcium folinate) [51] or [GA] (gemcitabine plus nab-paclitaxel) [52] regimens significantly prolonged the overall survival of patients with metastatic PDAC compared with gemcitabine monotherapy, implying that these combination regimens are more effective in killing tumor cells. In this regard, adding ICB drugs to intensive chemotherapy is speculated to further improve the prognosis of patients, mainly because the increased burden of neoantigens derived from lysed cancer cells can potentially improve anticancer immunity when these antigens are successfully presented by DCs to peripheral T cells [53]. When such T cells migrate into the tumor, they can recognize the cancer clones sharing the neoantigens and then kill these cancer cells [53]. Supporting this theory, recent data from several phase I and II trials indeed revealed that as a first-line therapy, chemotherapy plus ICB therapy had improved effectiveness compared with as a second- or later-line therapy in metastatic PDAC (Table 2). For example, gemcitabine plus tremelimumab achieved an ORR of 10.5% [54]. The [GA] regimen plus nivolumab (an anti-PD-1 drug) or plus pembrolizumab achieved ORRs ranging from 18% to 50% [55, 56]. More strikingly, when [GA] regimen was combined with durvalumab plus tremelimumab, the ORR was 73% [57]. In addition, an ORR of 80% was achieved when nivolumab was added to the regimen containing nab-paclitaxel, cisplatin, gemcitabine and paricalcitol [58]. These combinational strategies were tolerated by most enrolled patients. Therefore, although these trials had low patient numbers, their data at least provide new insights into the future management of metastatic PDAC by using chemotherapy plus ICB therapy in the first-line setting. Nevertheless, the prognostic value of this strategy in metastatic PDAC remains to be elucidated via randomized phase III trials.

Overall, the currently published data indicate an extremely low effectiveness of monotherapy by using ICI drugs or their combination with other conventional approaches, such as radiotherapy or chemotherapy, as second- or later-line therapies for metastatic PDAC (Table 2). In PDAC, only the MSI-H/dMMR phenotype is indicative of response to pembrolizumab. However, the prevalence of the MSI-H/dMMR phenotype in PDAC has been reported to be only 1% ~ 2% [59], but intriguingly, the MSI-H/dMMR phenotype was found to be strongly correlated with a high tumor mutational burden (TMB) and a wild-type KRAS and p53 molecular background [58]. Consequently, to achieve a breakthrough in the management of PDAC with KRAS mutation, a focus should be placed on eliminating the tumor cell- or stromal cell-induced barriers that counteract anticancer immunity. Recent studies in this field have revealed several strategies, such as adding an antiangiogenic drug [60], an anti-IL-6 antibody [61], an ATM inhibitor [62], a CD40 agonist [42], a CSF1R inhibitor [63], a YAP inhibitor plus a pan-RAF inhibitor [28], a CXCR4 inhibitor [41], a PARP inhibitor [64], a Listeria vaccine plus an anti-CD25 antibody [65], a FAK inhibitor [66], a CCR2 inhibitor [67], an IDO inhibitor plus the GM-CSF-conjugated whole-cell PDAC vaccine (GVAX) [68], or the combination of anti-PD-1 and anti-PD-L1 monoclonal antibodies [69], that have been confirmed to improve the immune milieu and the effectiveness of ICB therapy in preclinical models of PDAC. On these bases, some strategies using ICB therapy plus GVAX or other means, such as CXCR4 inhibition, CSF1R inhibition and CD40 blockade, have been designed to treat PDAC patients in clinical trials [39]. A few strategies have exhibited their effectiveness, such as the success of GVAX plus ipilimumab in prolonging the survival of PDAC patients [70].

Besides, a few small molecular compounds, such as AMG510, MRTX849, ARS-3248/JNJ-74699157 or LY3499446, have been designed to antagonize cancer cells carrying the KRAS^G12C mutation [71]. Among them, the data of AMG510 and MRTX849 are encouraging. For example, both in KRAS^G12C-mutated LUAC and CRAC models, basic experiments revealed the tumoricidal activity of AMG510 or MRTX849 both in vitro and in vivo [72, 73]; Likewise, administration of AMG510 or MRTX849 was confirmed to cause a significant shrinkage of tumors among patients with the KRAS^G12C-mutated LUAC, CRAC or PDAC [72-74]. Moreover, the tumoral immune milieu can be improved by using such KRAS^G12C inhibitors. In the model of mice bearing KRAS^G12C-muated CT-26 cell line-derived tumors, following AMG510 administration, T cells were found to significantly infiltrate into tumors [72]. Particularly, most of them were positive for CD8, and they presented a proliferating status upon AMG510 administration [72]. Mechanically, AMG510 administration could induce the upregulation of CXCL10 and CXCL11 by tumor cells, two crucial chemoattractant of T cells, thus causing an increasement of T cells in xenografted tumors [72]. Meanwhile, DCs including CD103⁺ cross-presenting pool and macrophages were found to increase their infiltration in xenografted tumors as well [72]. Functionally, CD103⁺ DCs are crucial for T cell priming and activation, while activated CD8⁺ T cells can produce IFN-γ, which enables tumor cells to increase their expression of MHC-I [72]. Thus, following AMG510 administration, the tumoral immune milieu was characterized by increased interferon signaling, antigen processing, chemokine production, cytotoxic activity and innate immune system stimulation [72]. Similar to AMG510, in the model of mice bearing KRAS^G12C-muated CT-26 cell line-derived tumors, MRTX849 administration was revealed to induce the polarization of TAMs from M2 to M1, the infiltration of DCs, B cells and tumoricidal T cells in tumors, as well as the reduction of MDSCs in tumors [75]. Therefore, either AMG510 or MRTX849 plus an anti-PD-1 antibody were demonstrated to cause a durable shrinkage of xenografted tumors with KRAS^G12C mutation [72, 75]. In fact, data associated with the potential of AMG510 or MRTX849 in shifting tumoral immune milieu from a suppressive to a tumoricidal state are mainly collected from the model of CRAC, rather than PDAC [72, 75]. In this regard, more efforts should be paid in the future to reveal whether KRAS^G12C inhibition can improve the tumoral immune milieu of PDAC, thus enabling the combination of KRAS^G12C inhibition and anti-PD-1 therapy to overcome the immunosuppression in PDAC. However, the frequency of KRAS^G12C mutation only accounts for less than 3% among all PDAC cases, whereas approximately 50% of PDAC cases have the missense form of G12D [9]. In fact, adaptive transfer of CD8⁺ T cells that react with KRAS^G12D-mutated tumor cells were demonstrated to be an effective approach in treating CRAC [76]. In order to benefit the majority of PDAC patients, drugs or new treatment strategies that target G12D missense mutation should deserve attention; In this scenario, the tumoricidal activity of newly developed approaches along with their potentials in improving tumoral immune milieu should be explored in the future.

5. Value of KRAS mutation for predicting cancer immune status in other adenocarcinomas

As mentioned above, PDAC, CRAC and LUAC are the top three cancers harboring a high prevalence of KRAS mutations [9] (Table 1). In CRAC, the prevalence of KRAS mutation is 44.7% [9]. As in PDAC, G12D is the most frequent missense mutation that causes consecutive activation of KRAS protein in CRAC [9] (Table 1). Among KRAS-mutant CRAC cases, 35% to 50% of them are reported to have concomitant inactivation in APC and p53 [77]. In mice bearing CRAC, the KRAS^G12D mutation was revealed to significantly increase the invasion and metastasis of cancer cells because conditional codeletion of Apc and Tp53 concomitant with KRAS^G12D mutation enabled primary and metastatic tumors to significantly upregulate the expression of the gene encoding TGF-β1, both a critical immunosuppressive cytokine [78] and a critical ligand of TGF-β/SMAD signaling that can dictate epithelial-mesenchymal transition (EMT) in CRAC cells [79]. Moreover, compared with patients with the wild-type RAS, CRAC patients harboring KRAS mutation generally have a poor prognosis [80].

However, the prevalence of the MSI-H/dMMR phenotype in CRAC is higher than that in PDAC. According to published data, the incidence of the MSI-H/dMMR phenotype in CRAC is 14% [81]. Currently, ICB therapy with pembrolizumab is recommended as the first-line therapy for metastatic CRAC with the MSI-H/dMMR phenotype, which has been confirmed as a reliable biomarker for predicting the outcome of ICB therapy by several lines of trial data [4, 82, 83]. Regardless, not all patients with this phenotype benefit from the ICB therapy [82, 83]. In the KEYNOTE-177 study, chemotherapy plus bevacizumab was still more effective than pembrolizumab monotherapy in prolonging the progression-free survival of patients with metastatic disease, the MSI-H/dMMR phenotype and KRAS mutation [84]. Conversely, those patients without KRAS mutation did benefit more from pembrolizumab than chemotherapy plus bevacizumab [84]. Hence, these results suggest that KRAS mutation can undermine the effectiveness of pembrolizumab even in the presence of the MSI-H/dMMR phenotype. Critically, KRAS mutation was revealed to be enriched in CRAC with the microsatellite stability (MSS) or proficient mismatch repair (pMMR) phenotype [81]. However, published data reveal that patients with CRAC with the MSS/pMMR phenotype respond poorly to ICB therapy alone [85].

Similar to its role in PDAC (Table 1), KRAS mutation in CRAC with the MSS/pMMR phenotype generally correlates with immune suppression in the tumor. To address this issue, a study evaluated the role of KRAS mutation in dictating the cancer immune status of CRAC tumors [86], which were mainly classified into four subgroups, namely, CMS1 (immune type), CMS2 (classical type), CMS3 (metabolic type) and CMS4 (mesenchymal type), according to which molecular pathways were enriched [81]. The results indicated that CMS2 or CMS3 tumors with KRAS mutation had a significantly reduced number of tumoricidal T cells compared with those without wild-type KRAS [86]. To explore the mechanism, experiments were performed in mice bearing CRAC with the KRAS^G12D mutation plus conditional depletion of Apc and Tp53, and this genetic alteration pattern was found to enable the tumors to have increased numbers of MDSCs but decreased numbers of CD4⁺ or CD8⁺ T cells compared with the pattern of conditional codeletion of Apc and Tp53 [87]. In detail, the KRAS^G12D mutation was able to activate ERK, which showed a negative relationship with the expression of gene encoding interferon-related factor 2 (IRF2) by tumor cells [86]. In return, IRF2 inactivation upregulated the expression of the gene encoding CXCL3, a chemokine that attracts MDSCs into tumors, thus impairing the expansion and IFN-γ-producing function of tumoricidal T cells [87]. Notably, KRAS mutation-related IRF2 inactivation was revealed to correlate with a poor response of CRAC patients to ICB therapy [87]. Conversely, in mice bearing CRAC with the KRAS^G12D mutation plus codeletion of Apc and Tp53, blocking CXCR2 on MDSCs improved the efficacy of anti-PD-1 therapy by increasing the number of CD8⁺ T cells but decreasing the number of Treg cells in tumors [87]. In fact, the CRAC tumors in these mice were revealed to resemble the CMS4 tumors in terms of some molecular signatures, such as the TGF-β/EMT signature [79]. As reported, the patients in the CMS4 subgroup commonly presented with rapid disease progression along with a poorer prognosis than the patients in other subgroups [81]. As such, KRAS mutation-induced immunosuppression potentially contributes to this process. In addition, CD8⁺ T cells that recognize the cancer cell clones carrying the KRAS^G12D mutation have been shown to exist in human CRAC tumors [88]. To our knowledge, the recognition of tumor antigens by tumoricidal T cells is as critical as having these cells infiltrate into tumors. Therefore, ICB therapy is speculated to improve the anticancer effect of T cells on KRAS-mutant CRAC.

In fact, KRAS-mutant CRAC still has several differences from PDAC in tumor biology (Table 1). As mentioned above, CRAC patients with the MSS/pMMR phenotype appear to be inherently refractory to ICB therapy [85]. Unlike in PDAC, the data from clinical trials, such as VOLTAGE (chemoradiation followed by 5 doses of nivolumab before radical surgery as a neoadjuvant therapy for locally advanced rectal cancer) [89], MEDETREME (FOLFOX regimen plus durvalumab and tremelimumab as a first-line therapy for metastatic CRAC) [90] and REGONIVO (regorafenib plus nivolumab as a third-line therapy for refractory CRAC) [91], have confirmed that patients with MSS/pMMR tumors could benefit from ICB therapy-based combinational strategies. Certainly, a portion of patients harboring KRAS mutations in their tumors are included in these studies, thus helping to elucidate the role of chemoradiation, duplet chemotherapy or molecule-targeted therapy in boosting the tumoricidal milieu. In addition to using conventional means, several new means have been developed. As documented, KRAS mutation-driven molecular alterations cause CMS3 tumor cells to have dysregulated glucose, glutamine, fatty acid and lipid metabolism [81, 92]. Targeting the metabolic abnormalities or blocking the downstream pathways affected by KRAS mutation, such as the MAPK and HIF-1-related pathways, has been shown to induce cancer cell death, potentially increasing the release of tumor antigens [92]. However, intriguingly, although KRAS-mutant CRAC cells have been revealed to consume glucose for their expansion, they are more resistant to glucose restriction than cells with wild-type KRAS [93]. This is another difference from PDAC cells, and murine pancreatic epithelial cells with the KRAS^G12D mutation with Lkb1 inactivation have been found to be sensitive to acute glucose restriction or glycolysis inhibition [24]. Consistent with this finding, LUAC cells in mice with homozygous KRAS^G12D/G12D mutation were more sensitive to glucose restriction than those with heterogeneous KRAS^G12D/wt mutation or KRAS^wt/wt, and a higher consumption of glucose occurred in LUAC cells with the KRAS^G12D/G12D mutation than in LUAC cells with other versions of KRAS [94]. Notably, G12C is the most common missense causing KRAS mutation in LUAC, with a prevalence of 30.9% in Western patients [9]. Nevertheless, KRAS mutation should not be regarded as a marker indicating immunosuppression in LUAC tumors because the immune milieu in KRAS-mutant LUAC tumors is heterogeneous. For example, the KRAS/LKB1 and KRAS/TP53 comutations enable LUAC patients to have dramatically different responses to ICB therapy because these two mutational patterns generally create a unique immune milieu in tumors (see details in Ref. [95]). Collectively, KRAS mutation can affect the cancer immune state in PDAC, CRAC and LUAC in different ways and contextures (Table 1).

6. Conclusion

The tumor milieu in PDAC is profoundly immunosuppressive, which renders monotherapy by using ICB drugs almost completely ineffective. Regarding the development of immunosuppression in PDAC, multiple factors are involved. Herein, KRAS mutation has been shown to be central in this process, because KRAS mutation can activate YAP-TAZ and JAK-STAT3 to elicit an immunosuppressive response, and this initial signaling can then be strengthened by coordination with TP53 inactivation and other genetic or molecular alterations. Overall, KRAS mutation generally correlates with tumor immunosuppression in PDAC. Nevertheless, in CRAC and LUAC, KRAS mutation can dictate the cancer immune environment in different ways. In these cancers, the immune milieu varies despite the commonality of KRAS mutation. This notion can be exemplified by KRAS-mutant LUAC, which exhibits a varied response to ICB therapy depending on the types of genetic alterations that cooccur with the KRAS mutation.

Abbreviation list

ADM：acinar-to-ductal metaplasia; APC: adenomatous polyposis coli protein; ATM: ataxia telaniectasia-mutated gene-coded protein; BRAC: breast cancer susceptibility gene; CAF: cancer-associated fibroblast; CCR: chemokine C-C motif receptor; CDKN2A: cyclin dependent kinase inhibitor 2A gene; CMS: consensus molecular subtype; CRAC: colorectal adenocarcinoma; CSF-1: macrophage-colony stimulating factor; CTLA-4: cytotoxic T lymphocyte-associated antigen-4; CXCL: chemokine C-X-C motif ligand; CXCR: chemokine C-X-C motif receptor; DC: dendritic cell; dMMR: deficient mismatch repair; EMT: epithelial-mesenchymal transition; FAK: focal adhesion kinase; GITR: glucocorticoid-induced tumor necrosis factor receptor; GM-CSF: granulocyte-macrophage colony stimulating factor; GVAX: GM-CSF-conjugated whole-cell PDAC vaccine; HIF-1: hypoxia-induced factor 1; HRAS: Harvey rat sarcoma viral oncogene homolog gene; H3K4: histone 3 lysine 4; ICB: immune checkpoint blockade; ICD: immunogenic cell death; IDO: indoleamine 2, 3-dioxygenase; IRF2: interferon-related factor 2; JAK: Janus kinase; KLF5: Krüppel-like factor 5; KLRG1: killer cell lectin like receptor G1; KRAS: Kristen rat sarcoma virus oncogene homolog gene; LKB1: liver kinase B1 gene; LUAC: lung adenocarcinoma; MAPK: mitogen-activated protein kinase; MCP-1: monocyte chemotactic protein 1; MDSC: myeloid-derived suppressive cell; MHC-I: major histocompatibility complex-I; MLL1: mixed lineage leukemia protein 1; MSI: microsatellite instability; MSS: microsatellite stability; MYC: MYC proto-oncogene; NRAS: neuroblastoma RAS viral oncogene homolog gene; NIX: encoded by BNIP3L, Bcl2/adenovirus E1B 19 KDa protein-interacting protein 3-like gene; Nrf2: nuclear-related factor 2; NTRK: neuro trophin receptor kinase gene; ORR: objective response rate; PARP: poly ADP-ribose polymerase; PanIN: pancreatic intraepithelial neoplasia; PDAC: pancreatic ductal adenocarcinoma; PD-1: programmed death-1; PD-L1: programmed death ligand 1; PIK3CA: phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha; PI3K: phosphatidylinositol 3-kinase; pMMR: proficient mismatch repair; PTEN: phosphatase and tensin homolog gene; RAF: RAF proto-oncogene; SBRT: stereotactic body radiation therapy; SMAD4: SMAD family member 4 gene; STAT3: signal transducers and activators of transcription 3; TAA: tumor-associated antigen; TAM: tumor-associated macrophage; TAZ: tafazzin; TGF-β: transforming growth factor-beta; Th: T helper cell; TP53: tumor protein P53 gene; Treg: regulatory T cell; TMB: tumor mutational burden; VEGF: vascular endothelial growth factor; WNT: “Wingless/Integrated”; YAP: yes-associated protein.

Conflicts of Interest: The author declared no conflict of interest.

Author Contributions: CP wrote this paper, GM and GY prepared the figure and tables.

Funding: This work was supported by National Natural Science Foundation of China [Grant No. 81874254], by Scientific and Technological Developing Scheme Foundation of Jilin Province [Grant No. 20200201400JC], and by Foundation of Scientific Research Planning Project of the 13^th Five-year Plan of Jilin Provincial Department of Education [Grant No. JJKH20201043KJ].

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