If Virchow and Ehrlich Had Dreamt Together: What the Future Holds for KRAS-Mutant Lung Cancer

Non-small-cell lung cancer (NSCLC) with Kirsten rat sarcoma (KRAS) mutations has notoriously challenged oncologists and researchers for three notable reasons: (1) the historical assumption that KRAS is “undruggable”, (2) the disease heterogeneity and (3) the shaping of the tumor microenvironment by KRAS downstream effector functions. Better insights into KRAS structural biochemistry allowed researchers to develop direct KRAS(G12C) inhibitors, which have shown early signs of clinical activity in NSCLC patients and have recently led to an FDA breakthrough designation for AMG-510. Following the approval of immune checkpoint inhibitors for PDL1-positive NSCLC, this could fuel yet another major paradigm shift in the treatment of advanced lung cancer. Here, we review advances in our understanding of the biology of direct KRAS inhibition and project future opportunities and challenges of dual KRAS and immune checkpoint inhibition. This strategy is supported by preclinical models which show that KRAS(G12C) inhibitors can turn some immunologically “cold” tumors into “hot” ones and therefore could benefit patients whose tumors harbor subtype-defining STK11/LKB1 co-mutations. Forty years after the discovery of KRAS as a transforming oncogene, we are on the verge of approval of the first KRAS-targeted drug combinations, thus therapeutically unifying Paul Ehrlich’s century-old “magic bullet” vision with Rudolf Virchow’s cancer inflammation theory.


Introduction
In 1900, the German Nobel laureate Paul Ehrlich suggested a concept of "magic bullets" ("Zauberkugeln") to specifically target invading microbes, a concept that was subsequently adapted to describe highly specific, oncogene-targeted cancer treatments [1]. More than 100 years later, non-small-cell lung cancer (NSCLC) with activating mutations of the Kirsten rat sarcoma (KRAS) oncogene-despite representing almost one-third of all lung cancer cases-remains a tumor entity for which no fully FDA-or EMA-approved oncogene-targeted therapies exist (for a broader overview of the frequencies of known oncogenic driver events in NSCLC we refer to [2][3][4][5][6]). Accordingly, affected patients still face a dismal prognosis [7][8][9][10]. In contrast to clinically approved oncogene-targeted therapies for various other malignancies, e.g., imatinib for BCR/ABL-positive chronic myeloid leukemia (CML) or EGFR and ALK inhibitors for EGFR-mutant and EML4/ALKrearranged NSCLC, respectively [11][12][13][14], the development of a "magic bullet" against mutant KRAS has remarkably challenged scientists and physicians alike because it had long been considered "undruggable" due to biochemistry constraints [15].
Another reason for the aggressive behavior and difficulty in treating KRAS-mutant lung cancer is its highly inflammatory phenotype [16]. The first to postulate a connection between chronic inflammation and cancer in the 19th century was Rudolf Virchow, a pathologist at Berlin's Charité hospital. He had observed the presence of leucocytes Table 1. Factors contributing to the disease heterogeneity of KRAS-mutant non-small-cell lung cancer (sources).

Mutant KRAS Proteins Orchestrate the Tumor Microenvironment
The abilities of cancer cells to promote local inflammation and to simultaneously escape immune-mediated elimination are important cancer hallmarks [76]. The tumor microenvironment (TME) represents an intricate ecosystem composed of multiple noncellular and cellular components including stroma and immune cells. Cancer cells actively shape the composition and functionality of the TME by direct cell-to-cell interactions and/or by chemokine secretion. Mutant KRAS proteins play a central role in this process. KRAS-dependent effector functions increase the expression of so-called immune checkpoints like programmed death ligand-1 (PDL1), which by binding to PD1 prevents T cells from killing cancer cells [77,78]. They also restrict cancer-cell-intrinsic MHC class II expression-essential for the recognition of cancer cells by T cells [79] and impair Tcell effector functions and antitumor immunity via cyto-/chemokine-mediated (e.g., IL-1, IL-6, IL-8, GM-CSF) induction of myeloid-derived suppressor cells (MDSCs), regulatory T cells and M2-differentiated tumor-associated macrophages (TAMs) [80][81][82][83][84][85][86][87][88][89] (Figure 1). Mutant KRAS also induces NF-kB and cooperates with MYC-two master regulators of inflammation and immunosuppression [90][91][92][93]. sion of stimulator of interferon genes (STING) and of immune-related expression signatures than wild-type tumors ("cold" TME) and therefore are typically checkpoint-inhibitorresistant [75,101]. Ongoing scientific efforts are seeking to decipher the mechanistic basis for this lack of ICI response in STK11/LKB1 co-mutated tumors with the aim of developing new strategies to turn "cold" tumors into "hot" ones and thus increase the benefit of immune checkpoint blockade for NSCLC patients with this prevalent genotype [102].

Efficacy of Direct KRAS(G12C) Inhibitors and Mechanisms of Resistance
The first evidence that inhibition of oncogenic KRAS is beneficial from a therapeutic viewpoint came from immunocompetent genetically engineered mouse models (GEMMs) of lung cancer in which lung tumors completely regressed upon genetic removal of mutant KRAS [24][25][26]. In the last decade, better biochemical insights into KRAS structural biology finally allowed researchers to develop direct KRAS(G12C) inhibitors like the preclinical compounds SML-8-73-1, compound 12 ("Shokat compound"), ARS-853 and ARS-1620, as well as the clinical compounds AMG-510 (Amgen), MRTX-849 (Mirati Immune checkpoint inhibitors (ICIs) block the PDL1-PD1 receptor interaction and thus can reinvigorate antitumor immune responses in some patients with so-called "hot" tumors. ICIs alone or in combination with chemotherapy have become standard-of-care treatment for NSCLC patients whose tumors express PDL1 and lack EGFR mutations or EML4/ALK rearrangements [94][95][96][97][98][99]. These immunologically "hot" tumors are characterized by the accumulation of proinflammatory cytokines, high PD-L1 expression and intratumoral accumulation of CD8+ tumor-infiltrating lymphocytes (TILs), which are required for ICIs to be effective [100]. In contrast, immunologically "cold" tumors are deprived of TILs. Interestingly, KRAS-mutant tumors with TP53 co-mutations are associated with a "hot" TME [73,74], whereas STK11/LKB1 co-mutated tumors exhibit lower expression of stimulator of interferon genes (STING) and of immune-related expression signatures than wild-type tumors ("cold" TME) and therefore are typically checkpoint-inhibitorresistant [75,101]. Ongoing scientific efforts are seeking to decipher the mechanistic basis for this lack of ICI response in STK11/LKB1 co-mutated tumors with the aim of developing new strategies to turn "cold" tumors into "hot" ones and thus increase the benefit of immune checkpoint blockade for NSCLC patients with this prevalent genotype [102].
The phase I/II CodeBreaK-100 trial (NCT03600883) investigating the clinically most advanced KRAS(G12C) inhibitor AMG-510 (international nonproprietary name (INN) sotorasib) reported a confirmed objective response rate (ORR) of~32% and a disease control rate (DCR) of~88% among lung cancer patients for the phase I trial part. Grade 3/4 toxicities and treatment discontinuation occurred in 11.6% and 7% of patients, respectively [111]. Based on this study, in December 2020, the FDA granted breakthrough therapy designation for sotorasib for patients with KRAS(G12C)-mutant, locally advanced or metastatic NSCLC following at least one prior systemic therapy. The phase II trial part with a data cutoff on 1 December 2020 and a median follow-up of 12.2 months validated the phase I results with a confirmed ORR of 37.1%, a DCR of 80.6% and a median duration of response of 10 months. The median progression-free survival was 6.8 months. Treatment-related adverse events (TRAEs) led to treatment discontinuation in 7.1% of patients. Most TRAEs were grade 1 and 2 and included diarrhea (31% any grade), nausea (19%), liver enzyme changes (15%) and fatigue (11%) (presented at the 2020 World Conference on Lung Cancer, 28-31 January 2021).
The median follow-up of the phase I/II KRYSTAL-1 trial (NCT03785249) for Mirati's MRTX-849 compound (INN adagrasib) was still relatively short (9.6 months) at the last study update presented at the 32nd EORTC-NCI-AACR Symposium (24-25 October 2020), but the data reported for NSCLC patients appear slightly better than those for AMG-510, although longer follow-up is still required [112]. Of the evaluable patients, 45% achieved a partial response, and the DCR was 96%. TRAEs led to treatment discontinuation in 4.5% of patients and most commonly included nausea (54%), diarrhea (48%), vomiting (34%), fatigue (28%) and increased liver enzymes (23%). The only commonly reported grade 3/4 TRAE was hyponatremia (3%) (the clinical efficacy parameters are summarized in Table 2). Adagrasib has a longer half-life of about 24 h compared to sotorasib (half-life 6.5 h), which allows for continuous drug exposure and sustained KRAS target inhibition. Adagrasib also penetrates the blood-brain barrier in murine models and showed efficacy in one patient with NSCLC and active brain metastases, an important feature for this highly metastatic disease with frequent brain/central nervous system manifestation. It is most likely, however, that allele-specific KRAS(G12C) inhibitors will be combined with other treatment modalities. Monotherapies presumably have limited long-term efficacy in terms of preventing adaptive resistance since (1) tumors with minor fractions of cancer cells that harbor non-G12C KRAS mutations will ultimately relapse due to selection of these subclones [113,114] and (2) several mechanisms have been proposed for how cancer cells can lose their KRAS dependency. Among others, these include YAP pathway activation [115] and increased KRAS(G12C) expression via EGFR or aurora kinase signaling [116]. Drug combinations can furthermore account for the fact that currently available KRAS(G12C) inhibitors only target the inactive, GDP-bound form of KRAS and thus rely on the residual intrinsic hydrolysis of GTP to revert KRAS into the GDP-bound state. This mechanism is vulnerable to adaptive responses that activate upstream signaling, e.g., via receptor tyrosine kinases (RTKs) like the ErbB family or FGFR [56,58,117]. These RTKs signal through SHP2 (encoded by PTPN11), increase the GTP-loaded "ON" form of KRAS and therefore reduce KRAS(G12C) inhibitor target engagement [118,119]. Inhibition of SHP2 reduces this conversion of GDP-into GTP-bound KRAS and overcomes adaptive resistance to MAPK-pathway-targeted agents including KRAS(G12C) inhibitors [120,121]. In a patient with NSCLC, a reduction of tumor volume has been observed when adagrasib was combined with the experimental SHP2 inhibitor TNO-155 (Novartis), and the corresponding phase I/II KRYSTAL-2 study is currently recruiting patients (NCT04330664). In another phase Ib clinical trial, the combination of sotorasib and the experimental SHP2 inhibitor RMC-4630 will be investigated [122,123]. Other drug combinations (adagrasib plus pan-ErbB inhibitor afatinib; ARS-1620 plus mTOR inhibitor (everolimus) or linsitinib) follow the same biological rationale of simultaneously inhibiting KRAS and the nucleotide exchange on KRAS via RTKs [124] (for a comprehensive overview of clinical trials investigating strategies to overcome resistance to drugs targeting the KRAS(G12C) mutation we refer to [125]).

Conclusions and Future Perspectives of Combination Therapeutic Approaches
More than a century after the pioneering scientific work of Rudolf Virchow and Paul Ehrlich [1,17,18], for non-small-cell lung cancer (NSCLC), the era of cancer immunotherapywhich harnesses the immune system to kill cancer cells-follows the era of groundbreaking discoveries in the field of oncogene-targeted therapies [19,20]. However, the progress made during the "targeted therapy revolution" for EGFR-mutant and EML4/ALK-rearranged lung cancers among other oncogene-addicted pulmonary malignancies (for a comprehensive overview of oncogene-directed therapies against NSCLC, e.g., with ROS1/NTRK, BRAF, MET and HER-2 aberrations, we refer to [126]) had largely spared KRAS-mutant NSCLC despite the anticipated efficacy of KRAS inhibitors for this highly prevalent but heterogeneous lung cancer subtype [11,12,14,[24][25][26]127] (Table 1). After overcoming biochemistry constraints to directly inhibit KRAS(G12C), the most frequent mutational subtype in NSCLC, the historical assumption of KRAS as being an "undruggable" target needs to be irrevocably discarded [103][104][105][106][107][108][109][110]. First reports from phase I/II clinical trials investigating the direct KRAS(G12C) inhibitors sotorasib and adagrasib are very impressive considering this notoriously hard-to-treat patient subgroup. Response rates are slightly inferior to those observed with other oncogene-targeted therapies (e.g., against mutant EGFR or rearranged EML4/ALK) in pretreated patients, and differences in response rates could be due to the heterogeneity of KRAS-mutant lung tumors with multiple DNA-damage-associated genomic alterations [128][129][130][131]. Additional KRAS(G12C) inhibitors are continuing to emerge for which clinical efficacy parameters have yet to be reported (JNJ-74699157/ARS-3248: NCT04006301; GDC-6036: NCT04449874); for others, the clinical development has been stopped due to unexpected toxicities (LY-3499446: NCT04165031). Currently ongoing (CodeBreaK-200 for AMG-510: NCT04303780) and future randomized phase III trials will ultimately show the true benefit of direct KRAS(G12C) inhibitors in untreated patients and presumably establish KRAS(G12C) inhibitors as the frontline treatment for KRAS(G12C)-mutant lung cancers (the current status of clinical development of KRAS(G12C) inhibitors is summarized in Table 2).
To induce deeper initial tumor regressions and to prevent the emergence of resistant cancer cell clones, multidrug combinations (e.g., KRAS(G12C) inhibitors with SHP2 and pan-ErbB inhibitors) are currently being clinically evaluated. Historically, drug combinations targeting KRAS-dependent downstream pathways (e.g., continuous MEK and PI3K inhibition) have been limited by toxicities [63,64], but KRAS(G12C) inhibitors avoid wild-type KRAS reactivity and therefore are less prone to off-target effects that are believed to disturb tissue homeostasis [127]. The lack of dose-limiting toxicities observed with sotorasib and adagrasib is encouraging and seems to make them ideal partners for combination treatment strategies, including those that incorporate immunotherapy.
Sensitivity to immune checkpoint inhibitors (ICIs) has been associated with a high tumor mutational burden (TMB) [132][133][134], and therefore, the smoking-related etiology of KRAS(G12C)-mutant NSCLC with a high mutational burden predestines affected patients for immunotherapy [44][45][46][69][70][71][72]135]. ICIs have been established as standard-ofcare treatment for NSCLC patients whose tumors express PDL1 and lack EGFR mutations or EML4/ALK rearrangements as a single agent or in combination with chemotherapy [94][95][96][97]134]. However, response rates to single-agent ICIs overall are modest, and strategies to overcome this limitation are urgently required. The clinical benefit of combined PD1/PDL1 and CTLA-4 inhibition remains controversial despite FDA approval of the ipilimumab plus pembrolizumab combination [136] and comes at the cost of an increased risk of serious immune-related adverse events compared to anti-PD-1 therapy alone [137].
Due to the important function of KRAS in reducing cancer cell immunogenicity and inducing local immunosuppression (Figure 1), KRAS(G12C) inhibitors were expected to have profound effects on the tumor microenvironment (TME). In KRAS-mutant NSCLC, the TME is frequently characterized by a paucity, lack and/or dysfunction of tumorinfiltrating leukocytes (TILs), especially in the presence of co-occurring mutations in STK11/LKB1 [41,75,101]. This immunologically "cold" TME impairs the efficacy of ICIs [100], and therefore, strategies to turn "cold" tumors into "hot" ones are urgently needed. Indeed, similarly to MEK and SHP2 inhibition, sotorasib and adagrasib induced a more proinflammatory and TIL-infiltrated TME in mouse models ("reconditioning" effect) [103,121,[138][139][140]. This translated into durable complete responses in combination with anti-PD-1 therapy. Mice that were cured with a combination of sotorasib and pembrolizumab subsequently rejected KRAS(G12C)-mutant CT26 tumors, suggesting that combining KRAS(G12C) inhibitors with immune checkpoint inhibitors could even drive an acquired immune response. Adaptive rather than innate immunity offers the greatest potential for durable, robust anticancer immune responses to prevent a tumor relapse and/or metastatic spread. However, these results from preclinical models have yet to be confirmed in human clinical trials. The combination of a KRAS(G12C) inhibitor and immunotherapy could specifically benefit those patients whose tumors harbor STK11/LKB1 co-mutations (~30% of KRAS(G12C)mutant NSCLC). These tumors are linked to poor outcomes with immunotherapy and platinum-based chemotherapy [75,141,142]. Even though numerous strategies aimed at bolstering immunity against STK11-mutant tumors are currently under investigation (e.g., dual immune checkpoint inhibition with nivolumab and ipilimumab [143]), a broader spectrum of efficacious therapeutic options is urgently needed. Exploratory correlative analyses from the KRYSTAL-1 (presented at the 32nd EORTC-NCI-AACR Symposium, 24-25 October 2020) and CodeBreaK 100 (presented at the 2020 World Conference on Lung Cancer, 28-31 January 2021) trials in this context suggest higher response rates for singleagent adagrasib (64% versus 45%) and sotorasib (50% versus 42%) among patients whose tumors also harbored an STK11/LKB1 co-mutation. Even though these early findings need to be confirmed in larger clinical trials that combine sotorasib (NCT03600883) and adagrasib (NCT04613596, KRYSTAL-7) with the anti-PD-1 antibody pembrolizumab, they give us a glimpse of the extraordinary potential of these KRAS-targeted agents for this historically difficult-to-treat patient subgroup.
Other strategies to boost the immune response against KRAS-mutant cancers include STING agonists (ADU-S100, MK-1454) [101,144,145], as well as CAR-T cells (adoptive T-cell transfer) [146] and mRNA vaccine technology [147]. For the latter, the current worldwide first use of mRNA vaccine technology to fight the COVID-19 pandemic [148,149] could boost its development and acceptance in the field of oncology. In an ongoing phase I trial (V941-001), Moderna and Merck are testing mRNA-5671 alone and in combination with pembrolizumab in patients with KRAS-mutant cancers. mRNA-5671 is designed to generate and present the four most prevalent KRAS mutations (G12C, G12D, G12V and G13C) as neoantigens in host cells to the immune system to drive a more robust T-cell response (no efficacy data are publicly available yet).
A major caveat we still face today is the fact that so far no specific inhibitors of non-G12C mutations have entered clinical trials. In NSCLC, these mutations represent more than 50% of all KRAS mutations. A potential first-in-class inhibitor of KRAS(G12D), MRTX-1133, is currently in preclinical development by Mirati (to the best of our knowledge, there are no publicly available data on this compound yet). In an alternative approach, the son of sevenless 1 (SOS1) protein, which determines the nucleotide exchange on KRAS, has gained much attention as a therapeutic target to inhibit all major G12D/V/C and G13D variants. Boehringer Ingelheim's BI-1701963 and Bayer's BAY-293 "pan-KRAS inhibitors" selectively inhibit the SOS1-KRAS interaction [150,151], but unfortunately, apoptosis induction and tumor regressions were only observed when this drug class was combined with a MEK inhibitor. Phase I dose-finding studies are currently recruiting patients with solid tumors (BI-1701963 plus trametinib: NCT04111458) or are planned (BI-1701963 plus adagrasib). Other strategies to target non-G12C mutations include so-called switch I/II pocket inhibitors like BI-2852, which bind with nanomolar affinities to the active and inactive forms of KRAS [152,153], or mutant-selective "tricomplex" inhibitors, which sterically block interactions between KRAS and effector proteins such as RAF [154]. KRAS-targeting monobodies [155] and intrabodies [156] further add to the spectrum of therapeutic approaches currently under investigation.
From an oncologist's perspective, the coming years in the field of KRAS-mutant NSCLC will be exciting and extremely laborious at the same time. Future clinical trials will have to teach us which drug combinations out of the multiple available therapeutic concepts (summarized in Figure 2) are the most efficacious ones and/or which treatment sequence is optimal from a tumor evolution perspective. The establishment of potent treatment predictors and systematic analysis of on-treatment longitudinal biopsies will hold the key to a better understanding of the biology of treatment resistance and guide clinical decision-making about rationally designed subsequent treatment combinations. To speed up the efficient development of drug combinations, the refinement of KRAS-mutant GEMMs to better recapitulate the genetic and immunologic complexity of human lung tumors with a high tumor mutational burden is absolutely desirable [157,158].
Despite some limitations, it is extremely exciting to see that more than a century after the groundbreaking scientific work of Paul Ehrlich and Rudolf Virchow, we are now on the verge of therapeutically unifying the concepts of both pioneers to harness synergistic effects between immune checkpoint inhibitors with "magic bullet" KRAS(G12C) inhibitors that have the potential to "recondition" the immunosuppressed tumor microenvironment. More than 40 years after the identification of RAS as a transforming oncogene, this approach will revolutionize paradigms for KRAS-mutant NSCLC. Therefore, after many discouraging therapeutic attempts in the past, we have every reason to look to the future with optimism.