Targeted Therapy for KRAS Mutant Non-Small Cell Lung Cancer

Ola Gaber¹, Ihab Eldesouki^1*, Mohamed Magdy², Mohamed Rahouma³, John Morris, C.¹ & Nagla Abdel Karim¹

¹Department of Hematology-Oncology, University of Cincinnati, USA
²Pediatric Oncology Department, Hospital, Cairo, Egypt
³Surgical Oncology Department, National Cancer Institute, Cairo University, Egypt

*Correspondence to: Dr. Ihab Eldesouki, Department of Hematology-Oncology, University of Cincinnati, USA.

Copyright © 2019 Dr. Ihab Eldesouki, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abbreviations
EGFR - Epidermal Growth Factor Receptor
EMT - Epithelial-Mesenchymal Transition
GEMMs - Genetically Engineered Mouse Models
GSIs - γ-Secretase Inhibitor
HGF - Hepatocyte Growth Factor
HOP - HSP Organizing Protein
HSP - Heat Shock Proteins
HSEs - Heat Shock Response Elements
IGF - Insulin-Like Growth Factor
KRAS - Kirsten Rat Sarcoma Viral Oncogene Homolog
MEK - Mitogen-Activated Protein Kinase Kinase
mTOR - Mammalian Target of Rapamycin
NSCLC - Non-Small Cell Lung Cancer
OS - Overall Survival
KRAS mutant - KRAS^mut
KRAS wild type - KRAS^wt
PD-1 - Programmed Cell Death-1
PDGFR - Platelet Derived Growth Factor Receptor
PD-1 - Programmed Cell Death Ligand-1
PFS - Progression Free Survival
RR - Response Rate
SD - Stable Disease
VEGF - Vascular Endothelial Growth Factor
AHA1 - Activator of HSP90 ATPase 1

Introduction
To understand the versatile role of KRAS mutations in carcinogenesis, a scrutinizing approach must be used to cellular pathways that control growth. KRAS is located on chromosome 12 (12p12.1), spanning ≈38 kilo-bases. K-Ras, which the protein product, is a 188 amino-acids with a molecular weight of 21.6 kDa. It is one of the RAS family of G-proteins, which are a group of intracellular guanine nucleotide binding proteins with GTPase activity. They are located on l the inner face of the plasma membrane. It functions as a binary molecular switch, when it is bound to GTP, it is in an active state, and when GDP-bound, it is considered in an inactive state. While in an active state, RAS-GTP complex activates several signal transduction cascades such as Raf-MEK-ERK, PI3K-AKT-mTOR and RalGDS-RalA/B pathways and TIAM1-RAC1 pathway. See figure 1. All play pivotal roles in cellular proliferation, apoptosis, survival and growth. Putting a RAS proteins in an active RAS-GTP conformation is achieved through multiple triggering signals from growth factors, like EGFR that favor a constitutive activation of KRAS, and also HGF and IGF when they interact with their receptors. The hydrolysis rate constant (kh) is of K-Ras is too low to be physiologically relevant, but specific “GTPase activating proteins” can increase hydrolysis by 100,000-fold. These are Guanine nucleotide exchange factors (GEFs), which decrease the affinity of RAS proteins for GDP and favor GTP binding resulting in RAS activation, while GTPaseactivating proteins (GAPs) accelerate the intrinsic GTPase activity to regulate the RAS cycle [1]. KRAS mutations are thus one of the driver mutations, and the gene is one of the principle oncogenes such as Raf, Myc, Src, Abl/Bcr and c-erbB-2. KRAS^mut were first described more than 40 years ago. They are usually missense mutations with an amino acid substitution at codons 12,13 or 61. Eighty percent of these mutations are in codon 12. The most common mutation is G–T transversion in 70% of tumors.

In 2005, Mascaux at al reported a meta-analysis to evaluate the prognostic value of KRAS^mut in lung cancer. Their study included 43 studies published from 1990 till 2003 with a total number of 5216 patients included in the meta-analysis. The authors concluded that RAS is a prognostic factor for lung cancer and KRAS was associated with poorer outcomes in NSCLC patients [2]. Another meta-analysis by Meng et al was published in 2013 reported similar results as they reported that KRASmut was associated with worse OS in NSCLC patients with HR of 1.45 (95% CI: 1.29-1.62). They also found that this is observed more the adenocarcinoma patients and interestingly enough in earlier stages [2].

Approximately one third of NSCLC cases carry activating point mutations in KRAS oncogene, mainly adenocarcinomas, and research has suggested that patients with KRASmut NSCLC fail to benefit from systemic chemotherapy compared to their KRASwt peers. Moreover, subgroup analysis has revealed that of these patients lack sensitizing EGFR mutations meaning the two mutations are mutually exclusive as well, thus targeted EGFR therapies cannot benefit these patients as well. Despite their high frequency in clinical settings and the vast amount of research done this field, KRASmut NSCLC still lack active treatment agents that target its specific oncogenic effects.

In this review, we searched Pubmed and Web of Science data bases from 2003 -2018 as well as www. ClinicalTrials.gov to find published and ongoing therapeutic trials specifically targeting KRAS^mut NSCLC.

Serine/Threonine Kinase Inhibitors
Phenformin, an analogue of the anti-diabetic drug metformin, and is an inhibitor of mitochondrial-induced apoptosis in cells defective in LKB1. LKB1, also called STK11, is the major upstream kinase that activates AMPK. About 30% of pulmonary adenocarcinomas have mutations that inactivates LKB1 so that the cells are less capable of sensing the metabolic effort.

In a preclinical study conducted in KRAS^mut-dependent mouse models of NSCLC, the antitumor effect of the drug phenformin was evaluated and the results showed selective responses to phenformin in tumors with both KRAS and Lkb1 mutations as a single agent. This was translated as prolonged survival in murine models [3].

In 2014, the same concept was investigated by a group of researchers using a panel of patient derived xenografts [4]. They used both metformin and phenformin to treat the xenografts and the tumors showed significant reduction in tumor volumes after 3 weeks of treatment. The tumors also maintained the response after caseation of the treatment. Lately, reports have emerged showing that Lkb1 mutations can be a cause for PD-1 resistance in KRAS^mut NSCLC adenocarcinoma [5]. Reportedly, combined KRAS and Lkb1 mutation had 7.4% RR to PD-1 inhibitors compared to 28.6% RR in KRAS^mut only cases. Also, lkb1 mutations were found to be associated with lack of PD-L1 expression on tumor cells.

mTOR is one of the major druggable targets in cancer since it falls at the interface of two major pathways PI3K and LKB1. Both pathways are a is activated in KRAS^mut tumors [6,7], and through which KRAS controls cellular growth and proliferation. Ridaforolimus is a small-molecule mTOR inhibitor and a serine/ threonine kinase inhibitor. Its effectiveness as a therapeutic agent in advanced KRAS^mut NSCLC stage IIIB/ IV who failed prior treatment, was studied in a randomized trial that included 79 patients [8]. The study reported overall RR of 1% at 8 weeks of initial treatment with ridaforolimus, however, the responders who received ridaforolimus after 8 weeks showed higher median PFS compared to placebo (4 months versus 2 months; HR 0.36, p=0.09). Overall survival from randomization was 18 months in the ridaforolimus arm and 5 months in the placebo group (HR 0.46, p = 0.09).

Heat Shock Protein (HSP) Inhibitors
HSPs are a family of proteins produced by cells in response to stresses. They act as chaperones stabilizing new proteins and ensuring their proper folding or by helping to refold damaged proteins [9,10]. HSP transcription occurs in response to heat shock factors (HSFs) that recognize the DNA enhancer sequences and heat shock response elements (HSEs) [11]. They recruit proteins through a complex of HSP70 and HSP40 co-chaperones, linked by an adapter molecule HOP (HSP organizing protein). The complex binds to a dimer of HSP90 protein bound to ADP. ADP-ATP exchange happens, that is activated by an adapter protein AHA1 (Activator of HSP90 ATPase 1), and allows a conformational change thus protecting the protein recruited, so that it can perform its functions. In this exchange of ATP, the recruited protein is coupled to ubiquitin and then shuttled to a proteasome [12]. Many proteins involved in cell proliferation and survival are clients of HSPs including receptor tyrosine kinases (RTKs), such as EGFR, ERBB2, KIT, MET, intracellular kinases, such as B-RAF, AKT and CDK4 factors transcript as p53, HIF1α or estrogen receptor, or protein as APAF1, BCL2 and telomerase. The HSP enable these proteins to withstand the hypoxic and acidic environment inside the tumor. They will even allow them to tolerate oncogenic genetic variations that would otherwise be lethal. HSPs are overexpressed in many solid tumors and hematological malignancies and they contribute to oncogenesis, having a positive effect on tumor cell survival and proliferation [13,14].

The activity of ganetespib (Synta Pharmaceuticals), a small molecule inhibitor of Hsp90 was examined in a panel of NSCLC cell lines harboring a wide range of KRAS mutations. In vitro, ganetespib demonstrated cytotoxicity through destabilization of the signaling effectors of activated K-RAS. Low concentrations of ganetespib in combination with MEK or PI3K/mTOR inhibitors resulted in enhanced cytotoxic compared to single agents in a subset of KRAS^mut cells. Consistent with this finding, the antitumor activity of ganetespib was increased by combining it with the irreversible PI3K/mTOR inhibitor, BEZ235 in human A549 xenografts in nude mice. At the molecular level, ganetespib abolished the activation of signaling feedback loops that are produced in response to inhibition of MEK and PI3K/mTOR, although this activity was not the sole determinant of the combinatorial advantage. Ganetespib in NSCLC cells harboring KRAS^mut enhanced the antimitotic effect of chemotherapy such as topoisomerase inhibitors and alkylating agents. These data highlight the promise of ganetespib as a single-agent or in combination of KRAS^mut-driven lung tumors [15].

A phase II trial, studied another HSP inhibitor, NVP-AUY922 (Institute for Cancer Research/Vernalis), and attempted to identify predictors of response [16]. The in vitro effects of NVP-AUY922 on proliferation and protein expression in NSCLC cell lines showed changes in gene expression induced by exposure to the investigated agent. These results were evaluated in xenograft models for tumor control and biological effects. NVP-AUY922 was found to be a potent inhibitor of in vitro growth in 41 NSCLC cell lines evaluated with an IC₅₀ <100 nM, IC₁₀₀ and in 36 of the 41 cell lines tested at <40 nM. This was in accordance with changes in the gene expression after exposure to NVP-AUY922 that is involved in wide range of cellular functions, including steadily decreasing dihydrofolate reductase (DHFR) expression. NVP-AUY922 slowed the growth of A549 (KRASmut) xenografts and decreased EGFR protein expression in H1975 xenografts harboring a sensitizing EGFR mutation. A phase Ib trial was published in October 2018. They recruited 9 patients with previously-treated metastatic non-squamous NSCLC were treated using a standard 3 + 3 design with pemetrexed at the standard 500mg/m² dose, plus: AUY922, however the study was closed due inability to obtain the investigational agent but the researchers reported a maximum tolerated dose of AUY922 with pemetrexed of 55mg/m². No reports about KRAS^mut were published [17].

MEK Inhibitors
Since KRAS mutations result in the activation of the RAF-MEK-ERK pathway, MEK inhibitors are thought as potential therapeutic agents for KRAS^mut NSCLC. MEK inhibitors are thought to confer their anti-cancer effects by stabilizing the pro-apoptotic BH3-only protein, BIM and BAD, and destabilization of MCL1 which a member of Bcl-2 family [18]. However, their effects in clinical setting were limited [19]. Recent approach of double hit combination therapy to increase the sensitivity to a certain agent [20,21] has led researchers to investigate the possibility of adding a Bcl-2 family inhibitor can increase the efficacy of a MEK inhibitors in this subset of patients. A group of researchers treated a panel of 53 NSCLC and pancreatic cancer cell lines with a combination of navitoclax (ABT-263, Abbott Laboratories), a Bcl-2/BclxL (BCL2/BCL2L1) antagonist, and a novel MEK inhibitor, G-963. The combination yielded a synergistic effect in the majority of lines, with more cell lines harboring KRAS mutations in the highly synergistic group. Cells exposed to G-963 were found to be arrested in G1 phase with only a small fraction actually undergoing apoptosis. The addition of navitoclax to G-963 greatly increased the percentage of cells that underwent apoptosis with no change observed in the cell cycle arrest part. Same combination was more effective than either single-agent in the KRAS^mut H2122 xenograft model; BIM stabilization was observed in the tumors which is consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor, GDC-0941, to this treatment combination increased cell killing compared to double or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways that can be improved by combination with a Bcl-2 family inhibitor [22]. Accordingly, other MEK inhibitors are currently under investigation, including Salirasib [23], Tivantinib [24] (MET inhibitor), trametinib (which showed a good activity in metastatic melanoma with GNQ mutations) [20] and others [25-27]. All reported initial response to the investigational agents, however, they all await multicenter trails to be used in clinical practice.

Secretory Phospholipase A₂
The approach to investigate early on biomarkers that have screening, early diagnostic, predictive and prognostic value became the mainstay for cancer research. The aim is to find cost effective, less time consuming, specific markers to determine a predict tumor behavior and plan the treatment accordingly [20,28]. One of these molecules is the isoenzyme group IIa secretory phospholipase A2(sPLA₂IIa). In one study, sPLA2IIa was found to have significantly elevated levels in lung cancer patients [29], and hence this enzyme is involved in tumor promoting eicosanoids it was rendered as a potential biomarker. Another group investigated its correlation with KRAS^mut tumors in lung [30]. They found that sPLA₂IIa expression correlates with increasing stage of KRAS^mut lung tumors. Since the same group reported sPLA₂IIa inhibitors cytotoxic effect correlate with sPLA₂IIa expression, it was hypothesized that sPLA₂IIa modulates lung cancer cell growth in KRAS^mut cells. This opens the field for a new druggable target in this subset of patients, especially advanced cases.

Antiangiogenic Agents
Sorafenib, a small molecule inhibitor of several receptor tyrosine kinases such as VEGFR2, PDGFR, KIT as well as pathways such as Raf, was also investigated in several studies [31-34]. In a phase II trial, 59 patients with KRAS^mut lung cancer who previously received platinum-based therapy and stage IIb or IV were treated with sorafenib. The median PFS was 2.3 months median overall survival (OS) was 5.3 months. Patients with a prediction of good prognosis according to VeriStrat serum proteomics assay showed a significantly superior PFS [HR, 1.4; 95% confidence interval (CI), 1.0-1.9] but not OS (HR, 1.3; 95% CI, 0.9-1.7). Another, phase II trial, the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE-1), the disease control rate for patients treated with sorafenib was 61% in patients with KRAS mutations versus 56% in those with wild type KRAS. And a third phase II study of sorafenib carried out in 37 previously treated patients with non-small cell lung cancer were 11 patients (32%) had KRAS mutations. The disease control rate observed in these patients was not significantly different at 60% versus 71% in those with KRAS wild-type tumors (p=0.69).

Hedgehog Pathway Inhibitors
The hedgehog pathway is a group of intracellular signaling family of proteins. They were first isolated in the early 1990s and they are known to play a fundamental role in embryonic development including regulation of cellular proliferation, differentiation, epithelial to mesenchymal transition (EMT) and stem cell renewal and differentiation [35,36]. In adults, loss of some components of hedgehog functions was found in some hereditary diseases, however, in cancer overexpression of these proteins was reported, such as basal cell carcinoma, colon, lung, and pancreas cancer [37-39]. Also, hedgehog signaling has been implicated in tumor recurrence by promoting the survival of tumorigenic precursors and their effects on the tumorassociated stroma during advanced NSCLC [40,41]. One report evaluated the effects of the combination of an inhibitor targeting the hedgehog pathway combined with radiation in NSCLC cell lines in vitro using an A549 xenograft model [42]. They looked at tumor growth, proliferation, apoptosis, and gene expression changes after radiotherapy and concomitant HhAntag, a hedgehog signaling antagonist. In a transgenic mouse model of KRAS^mut (G12D)-induced lung adenocarcinoma and induced TWIST1, they evaluated the tumor response using micro-computerized tomography (CT). They found that in 4 human lung cancer cell lines, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. This was associated with down-regulation of the hedgehog pathway in stromal cells resulting in cellular apoptosis.

A USA group of investigators published preliminary data in 2018 based on analysis of human lung cancer xenografts and preclinical Genetically Engineered Mouse Models (GEMMs) of KRAS^mut NSCLC. They found that “the epithelial-mesenchymal transition (EMT) status of tumor cells is critical to their therapeutic response to MEK inhibitors”, with the epithelial state show high sensitivity to MEK inhibitors and the mesenchymal state results resistance to therapy [43]. This point can be an entry point to hedgehog inhibitor role in KRAS^mut NSCLC.

Notch Pathway Inhibitors
Notch pathway is an evolutionary conserved signaling pathway. It mediates short-range signaling cell to cell intercommunications [44]. Notch receptor ligands are transmembrane proteins [45] that result in NOTCH cleavage by γ-secretase complex [44]. One of the KRAS oncogenic mediators is Discoidin Domain Receptor 1 (Ddr1) gene which encodes a receptor protein kinase [46]. Ddr1 activates cell proliferation through a NOTCH dependent pathway [47]. So, the role of this pathway in the generation and maintenance of KRAS^mut (G12V)-driven NSCLCs was studied using γ-secretase inhibitor (GSIs) [48]. The investigators were able to show that γ-secretase is essential for NSCLC development, and pharmacological treatment of mice carrying autochthonous NSCLCs with a GSI inhibited cancer growth. In another research, the investigators utilized dual treatment of TP53-mutant patient-derived lung xenografts (PDX) with Ddr1 and NOTCH inhibitors, KRAS^mut showed regression with efficacy comparable to standard chemotherapy [49].

SRC-TKI Inhibitors
Both Src protein levels and Src protein kinase activity, are frequently elevated in human cancers in comparison to adjacent normal tissues. Moreover, these levels appear to increase with the stage of disease [50]. And in 2016, a study by Zhou et al has reported that KRAS^mut NSCLC patients had SRC expression levels that is as comparable to non-mutant cases [51]. Pemetrexed is an antifolate agent that is widely used to treat NSCLC. Resistance to pemetrexed is associated with thymidylate synthase (TYMS) overexpression. Using a Src inhibitor was reported to improve response to pemetrexed in these cases and even revert the developed resistance. The researchers noticed a positive correlation between TYMS and Src transcript levels. So, a combination of Src inhibitors with pemetrexed were attempted in vitro as means to improve the patients’ response to pemetrexed [21,52]. However, this hypothesis requires a thorough investigation of TYMS and Src overexpression, protein level and activity and KRAS mutations in NSCLC.

Farnesyltransferase Inhibitors and Trans-Farnesylthiosalicylic Acid
As mentioned before, RAS proteins are membrane-bound intracellular GTPases. In fact, to exert their full signaling effects they need to remain in this membrane-bound status. The enzyme farnesyl protein transferase initiates binding of RAS protein to the membrane and hence using farnesyl protein transferase inhibitors in the human colon carcinoma cell line DLD-1. However, H-Ras only showed response to treatment, meanwhile K-Ras and N-Ras remained in a membrane -bound state [53]. Salirasib is an s-trans, trans-farnesylthiosalicylic acid which was found to prevent membrane binding in all forms of Ras proteins in contrast to farnesyl protein transferase inhibitors [53,54]. Salirasib was investigated in a phase I trial and showed good tolerance to treatment [55]. Recently, results from another phase I study in japanese patients was published. The agent was used as oral twice daily dose in 21 solid tumor patients who failed previous treatments. Only 4 cases tested KRAS^mut positive, however, they achieved a remarkable median PFS of 227 days [56].

Immunotherapy and Other Promising Approaches
Programmed death 1 (PD-1) is a key immune-checkpoint receptor expressed by activated T-cells and mediates immunosuppression within the tumor microenvironment. Overexpression of the PD-1 ligand (PD-L1) has been reported in NSCLC [57]. Since anti-PD-1 and PD-L1 been used in NSCLC therapy, the estimated 5-year OS rate was 16% for patients with pretreated, advanced NSCLC [58]. Thorough research showed that many NSCLC cell-lines with KRAS, EGFR and BRAF mutations, and ALK or RET rearrangements have high levels of PD-L1 expression, randomized phase III trials results indicated that patients with EGFR mutations exhibit less efficacy of anti-PD-1/PD-L1 treatment than those with wild-type EGFR. Meanwhile, KRASmut were found to be more likely to be predictors of favorable outcomes [59,60]. These results were particularly true for nivolumab, but not for atezolizumab [61,62]. Even though these results are very promising, the molecular bases are not well understood, yet. A group of researchers tried knocking down KRAS or ERK2 using siRNAs or ERK2 inhibitors, and this resulted in decreased PD-L1 expression in KRASmut NSCLC with PD-L1 overexpression. Same effect was observed with MEK inhibitors [63-65].

K-Ras(G12C) Inhibitors
In 2013, Ostrem et al published a report about small molecules that can irreversibly bind to K-Ras(G12C) mutant form only [66]. K-Ras(G12C) is a form of mutant K-Ras protein in which a transversion at 34th nucleotide occurs and glycine at residue 12 is replaced with cysteine (G12C) [67]. The K-Ras(G12C) inhibitors bind to the mutant cysteine thus sparing wild forms. After binding, they result in disruption in the K-Ras protein crystal structure so that its affinity to GDP becomes higher than GTP [66]. Later other reports emerged about different forms of K-Ras(G12C) inhibitors such as SML-8-73-1 which binds in a manner similar to GDP [68], and SML-10-70-1 which is a partially caged version of SML-8-73-1, which allows better transportation across cell membranes [69]. The results from these agents are very promising with high specificity to tumor tissue since the drug has minimal affinity to wild type K-Ras and good potency.

Discussion
Lung cancer is known for its high incidence, prevalence and lethality, both due to disease or other condition based complications [70,71]. However, it is known for its diverse subtypes that pose a challenge to current clinical practices and resistance to therapies [28,72-74]. NSCLC represent the majority of this group (80%) with KRAS mutations identified in in 20-30% of adenocarcinomas and less so in squamous-cell carcinomas of the lung [70]. This subset of patients was identified as worse responders to current therapies [75], and despite the fact that KRAS is a driver mutation in NSCLC with relatively well described downstream signaling effects, no definite therapy has been developed targeting its effects. Due to its diverse effects on the signaling pathways, KRAS^mut NSCLC are most likely to be targeted by combination therapies. However, due to the complexity of the downstream signals affect by mutant K-Ras active form, the aforementioned arsenal of therapies needs to be addressed in trials to assess efficacy, responses and safety. Meanwhile, approved agents can be preferentially used by oncologists in clinics according to current guidelines, but with attention to agents who shows anti-KRAS activity in preclinical studies. This approach will put PD-1/PD-L1 inhibitors early in the list, so as sorafenib, ridaforolimus, busotinib and salirasib among others mentioned in our review.

Stepping into a targeted combination therapy is plausible to be the focus of the preclinical studies. Lung cancer treatment have not seen much improvements in the standard of care treatments for the past 50 years, till the introduction of PD-1/PD-L1 inhibitors. This requires a better understanding of downstream signaling pathways that are affected by KRAS. So, more focus should be invested in this area.

Conflicts of Interests
The article is free from any conflict of interests.

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