A novel anticancer natural product SP09 selectively targets KRAS-mutant NSCLC through LKB1/AMPK/mTOR modulation: implications for novel therapeutic development

Lung cancer is the most common form of cancer worldwide and the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, and KRAS mutation occurs in 25-30% of NSCLC. However, to date, direct therapeutic targeting of KRAS has not been achieved. Our approach to developing a KRAS mutant NSCLC therapeutic agent was to identify a selective novel target involved in modulating signaling proteins in the RAS pathway without impacting healthy cells. Numerous studies have shown that glutathione S-transferase P (GSTP) is strongly upregulated in many cancer types, specifically lung, colon, and pancreas. Besides being a well-known Phase II detoxification enzyme, GSTP is also known to influence cell signaling through interaction with MAP kinases such as JNK and c-jun. We therefore hypothesized that GSTP knockdown (KD) could be an effective therapeutic approach to treat KRAS mutant tumors. To test this hypothesis, we designed a series of novel GSTP siRNAs and identified our lead candidate, a highly potent and selective GSTP siRNA (NDT-05-1040) to specifically target and downregulate GSTP protein. A panel of KRAS mutant NSCLC cell lines was utilized to determine the GSTP KD potency and antiproliferative activity of NDT-05-1040. Western blot analysis and co-immunoprecipitation (co-IP) were conducted to identify the proteins that were regulated by GSTP in the proliferation and apoptosis pathways. These studies demonstrated that NDT-05-1040 is a very potent and selective GSTP siRNA. Furthermore, transfection of NDT-05-1040 led to significant growth inhibition and induced apoptosis in NSCLC cells without affecting normal cell viability. Western blot analysis showed that NDT-05-1040 effectively decreased the phosphorylation of CRAF, ERK, MEK, in the MAP kinase pathway. Interestingly, with NDT-05-1040 treatment, the phosphorylation of Akt and mTOR in the PI3K pathway was also decreased. Moreover, the activity of p-JNK, PUMA, caspase-3/7, and p53 in the KRAS mutant cells was upregulated by NDT-05-1040. Co-IP experiments demonstrated that GSTP formed a complex with both p-CRAF and JNK. In summary, GSTP KD by NDT-05-1040 targeted the RAS/MEK/ERK, Akt, mTOR and apoptosis pathways. This selective multipronged attack on the growth and survival pathways of cancer cells makes it a compelling target and could potentially serve as a novel therapeutic agent for KRAS mutant NSCLC. Citation Format: Cima Cina, Jens Harborth, Zhihong O'Brien, Nish Beltran-Raygoza, Jung-kang Jin, Jessica Xu, Sang Jun Lee, Kwok Tsang, Jiping Yao, Roger Adami, Sonya Zabludoff, Wenbin Ying. A novel glutathione S-transferase P (GSTP) siRNA (NDT-05-1040) for the treatment of KRAS-driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5918.

A large fraction of non-small cell lung cancers (NSCLC) have mutant KRAS, which is associated with poor response to current cytotoxic therapy and a poor prognosis. Although the KRAS signaling pathway has been well characterized, no current therapies target this critical oncogene. Several studies have demonstrated that bypass of senescence in Kras-mediated adenocarcinoma mouse models is essential for tumorigenesis. Therefore, activation of senescence in KRAS mutant NSCLC may be an effective therapeutic strategy. We recently demonstrated that the basic helix loop helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in transgenic mouse models and that inhibition of Twist1 in these models led to activation of Kras-induced senescence and tumor stasis. In the current study, we examine the role of TWIST1 in KRAS mutant human NSCLC. Silencing of TWIST1 in multiple KRAS mutant NSCLC cell lines resulted in dramatic growth inhibition and either reactivation of oncogene-induced senescence or in some cases, apoptosis. Similar effects were also observed in four KRAS wild type lines, including cell lines with key driver mutations including a cell line with an activating EGFR mutation and a cell line with c-Met amplification. Gene set enrichment analysis of NSCLC cell lines after silencing of TWIST1 revealed a striking cell cycle arrest gene signature. Growth inhibition by silencing of TWIST1 was independent of p53 or Rb/p16 mutational status. Furthermore, activation of oncogene-induced senescence by TWIST1 silencing did not require previously defined mediators of senescence, p21 and p27, nor could this phenotype be rescued by overexpression of SKP2. To extend these observations in vivo, TWIST1 was silenced in both KRAS mutant and wildtype cell lines and these cells were implanted in NOD-SCID mice to assess tumor formation. Interestingly, silencing of TWIST1 in xenograft models preferentially inhibited KRAS mutant tumor formation suggesting that TWIST1 plays a critical in mediating KRAS tumorigenesis. Finally, inducible silencing of TWIST1 resulted in significant growth inhibition of established xenograft KRAS mutant tumors. Together these findings suggest TWIST1 is essential for the establishment and maintenance of KRAS mutant NSCLC tumors and silencing of TWIST1 in KRAS mutant NSCLC represents a novel and promising therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2954. doi:1538-7445.AM2012-2954

KRAS and EGFR mutations are common oncogenic drivers in NSCLC. Osimertinib is a first line therapy for EGFR exon 19 and 21 mutant NSCLC. Sotorasib and Adagrasib are a second line therapy for KRAS G12C mutant NSCLC. Single agent inhibitors targeting mutant EGFR or KRAS develop drug resistance leading to relapsed/refractory NSCLC. SF2523 is a dual PI3K-bromodomain-containing protein 4 (BRD4) small molecule inhibitor being evaluated to mitigate KRAS or EGFR inhibitor induced drug resistance. Co-inhibition of PI3K/BRD4 blocks MYC expression, activation and enhances MYC degradation leading to inhibition of cancer growth and metastasis. We investigated SF2523 in EGFR mutant (HCC2935 and HCC827) and KRAS mutant (NCI H2030 & NCI H23) NSCLC cells lines with single agent or combination with Osimertinib or Sotorasib for anti-tumor activity, respectively. SF2523 IC50 in EGFR mutated NSCLC cells ranged from 90-230nM, while in KRAS mutated NSCLC cells ranged from 144-217nM versus Buparlisib plus JQ1 in the μM range. SF2523 plus Osimertinib decreased the IC50 to 1.2-1.4nM and SF2523 plus Sotorasib 1.6-13nM, respectively. In contrast, combinations of SF2523 plus Nindetinib (VEGFR inhibitor) or Rapamycin (mTOR inhibitor) were additive in potency with EGFR and KRAS mutant NSCLC cell lines. In KRAS mutant NSCLC treatment with SF2523 or Sotorasib increased PTEN mRNA expression and the combination therapy was additive. In EGFR mutant NSCLC treatment with SF2523 or Osimertinib or in combination decreased MYC, PIK3CA and AKT1, respectively. In a SCID H2030 KRAS G12C mouse xenograft model, SF2523 at a dose of 150 mg/kg was well tolerated without weight loss with tumor growth inhibition of >60% at 6 weeks. In conclusion, our results demonstrate SF2523 is active alone and is synergistic with Osimertinib or Sotorasib in inhibiting EGFR or KRAS mutant NSCLC cell lines and is active as a single agent in a KRAS G12C mutated H2030 CDX NSCLC mouse model. Citation Format: Baskaran Subramani, Patrick Joseph Conway, Kun Zhang, Guillermo A. Morales, Joseph R. Garlich, Daruka Mahadevan. SF2523, a dual PI3K-BRD4 inhibitor, enhances KRAS (G12C) or EGFR (exon 19/21) mutant targeted inhibitors in non-small-cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 543.

Background: Lung cancer is the deadliest cancer in both men and women. About 85% of lung cancers are non-small cell lung cancer (NSCLC) and almost 25% of all types of NSCLC contain KRAS mutations, which remain as an undruggable challenge. Doublecortin-like kinase 1 (DCLK1) regulate many key oncogenes including KRAS in various solid cancers is overexpressed in lung cancer, raising the possibility to selectively target DCLK1 expression to combat the undruggable KRAS mutant NSCLC and its drug resistance. Experimental Procedure: H460 and A549 (KRAS mutant NSCLC cell lines) and H1299 (WT KRAS NSCLC cell line) were used. Cisplatin resistance KRAS mutant NSCLC cells (CR-A549) were generated. Transfection of siRNA against DCLK1 in NSCLC cells and CR-A549 cells were carried out. Tumor cell apoptosis, DNA damage, DNA damage response (DDR) and tumor cell self-renewal were assessed. Protein and mRNA expressions by western-blot and RT-PCR, apoptosis by FACS, cell death by COMET assay and self-renewal by clonogenic assay. Results: Here, we report that increased expression of DCLK1 is the key to develop chemoresistance and self-renewal in KRAS mutant NSCLC. We observed increased immunostaining for DCLK1 in patient lung adenocarcinoma compared to normal lung tissue. We observed an increased expression of DCLK1 protein and mRNA in KRAS mutant NSCLC cells compared to WT KRAS NSCLC cells. To investigate the role of DCLK1 in KRAS mutant NSCLC tumor progression, we knockdown DCLK1 in KRAS mutant NSCLC cells, which resulted in reduced cell proliferation/survival and self-renewal. We generated KRAS mutant NSCLC cisplatin resistance cells (CR-A549) and found that CR-A549 cells acquired higher DCLK1 expression with enhanced self-renewal capacity and display ~9fold higher IC50 for cisplatin treatment compared to parental cells. However, silencing DCLK1 in CR-A549 cells reversed the tumor cell resistance to cisplatin and increased cell death and reduced self-renewal. To further investigate mechanistically, we examine the expression of DDR signaling in CR-A549 cells. We observed an increased expression of ATR-DDR in CR-A549 cells. Furthermore, DCLK1 knockdown reduced the expression of ATR and treatment of siATR plus cisplatin (5µM) to CR-A549 cells demonstrated similar effects observed in the treatment of siDCLK1 plus cisplatin (5uM), which resulted in increased sensitivity of CR-A549 cells to cisplatin, and thus increased cell death and complete abrogation of self-renewal capacity, suggesting that DCLK1 mediated cisplatin resistance occurs via ATR-DDR dependent mechanism. Conclusion: Our data demonstrate that the increased expression of DCLK1 is associated with enhanced cancer stem cell-like features, ATR-DDR signaling and chemoresistance in KRAS mutant NSCLC. Targeting DCLK1 alone or in combination with cisplatin may represent a novel therapeutic strategy for the effective treatment of undruggable KRAS mutant NSCLC. Citation Format: Courtney Houchen, Janani Panneerselvam, Priyanga Mohandoss, Nathaniel Weygant, Randal May, Dongfeng Qu, Naushad Ali, Timothy Wang, Chinthalapally Rao, Michael Bronze, Parthasarathy Chandrakesan. DCLK1 regulates ATR-DNA damage response for KRAS mutant lung cancer drug resistance and stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2119.

Background: Lung cancer is the deadliest cancer in both men and women. About 85% of lung cancers are non-small cell lung cancer (NSCLC) and almost 25% of all types of NSCLC contain KRAS mutations, which remain as an undruggable challenge. Doublecortin-like kinase 1 (DCLK1) regulate many key oncogenes including KRAS in various solid cancers is overexpressed in lung cancer, raising the possibility to selectively target DCLK1 expression to combat the undruggable KRAS mutant NSCLC and its drug resistance. Experimental Procedure: H460 and A549 (KRAS mutant NSCLC cell lines) and H1299 (WT KRAS NSCLC cell line) were used. Cisplatin resistance KRAS mutant NSCLC cells (CR-A549) were generated. Transfection of siRNA against DCLK1 in NSCLC cells and CR-A549 cells were carried out. Tumor cell apoptosis, DNA damage, DNA damage response (DDR) and tumor cell self-renewal were assessed. Protein and mRNA expressions by western-blot and RT-PCR, apoptosis by FACS, cell death by COMET assay and self-renewal by clonogenic assay. Results: Here, we report that increased expression of DCLK1 is the key to develop chemoresistance and self-renewal in KRAS mutant NSCLC. We observed increased immunostaining for DCLK1 in patient lung adenocarcinoma compared to normal lung tissue. We observed an increased expression of DCLK1 protein and mRNA in KRAS mutant NSCLC cells compared to WT KRAS NSCLC cells. To investigate the role of DCLK1 in KRAS mutant NSCLC tumor progression, we knockdown DCLK1 in KRAS mutant NSCLC cells, which resulted in reduced cell proliferation/survival and self-renewal. We generated KRAS mutant NSCLC cisplatin resistance cells (CR-A549) and found that CR-A549 cells acquired higher DCLK1 expression with enhanced self-renewal capacity and display ~9fold higher IC50 for cisplatin treatment compared to parental cells. However, silencing DCLK1 in CR-A549 cells reversed the tumor cell resistance to cisplatin and increased cell death and reduced self-renewal. To further investigate mechanistically, we examine the expression of DDR signaling in CR-A549 cells. We observed an increased expression of ATR-DDR in CR-A549 cells. Furthermore, DCLK1 knockdown reduced the expression of ATR and treatment of siATR plus cisplatin (5µM) to CR-A549 cells demonstrated similar effects observed in the treatment of siDCLK1 plus cisplatin (5uM), which resulted in increased sensitivity of CR-A549 cells to cisplatin, and thus increased cell death and complete abrogation of self-renewal capacity, suggesting that DCLK1 mediated cisplatin resistance occurs via ATR-DDR dependent mechanism. Conclusion: Our data demonstrate that the increased expression of DCLK1 is associated with enhanced cancer stem cell-like features, ATR-DDR signaling and chemoresistance in KRAS mutant NSCLC. Targeting DCLK1 alone or in combination with cisplatin may represent a novel therapeutic strategy for the effective treatment of undruggable KRAS mutant NSCLC. Citation Format: Courtney Houchen, Janani Panneerselvam, Priyanga Mohandoss, Nathaniel Weygant, Randal May, Dongfeng Qu, Naushad Ali, Timothy Wang, Chinthalapally Rao, Michael Bronze, Parthasarathy Chandrakesan. DCLK1 regulates ATR-DNA damage response for KRAS mutant lung cancer drug resistance and stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2119.

KRAS mutations are one of the most common driver mutations in non-small-cell lung cancer (NSCLC) and finding druggable target molecules to inhibit oncogenic KRAS signaling is a significant challenge in NSCLC therapy. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in both KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the current study, we found that EREG is overexpressed in NSCLCs harboring KRAS, BRAF or EGFR mutations compared with NSCLCs with wild-type KRAS/BRAF/EGFR. Small interfering RNAs (siRNAs) targeting mutant KRAS, but not an siRNA targeting wild-type KRAS, significantly reduced EREG expression in KRAS-mutant and EREG-overexpressing NSCLC cell lines. In these cell lines, EREG expression was downregulated by MEK and ERK inhibitors. Importantly, EREG expression significantly correlated with KRAS expression or KRAS copy number in KRAS-mutant NSCLC cell lines. Further expression analysis using 89 NSCLC specimens showed that EREG was predominantly expressed in NSCLCs with pleural involvement, lymphatic permeation or vascular invasion and in KRAS-mutant adenocarcinomas. In addition, multivariate analysis revealed that EREG expression is an independent prognostic marker and EREG overexpression in combination with KRAS mutations was associated with an unfavorable prognosis for lung adenocarcinoma patients. In KRAS-mutant and EREG overexpressing NSCLC cells, siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis. Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

KRAS gene mutation is linked to poor prognosis and resistance to therapeutics in non-small cell lung cancer (NSCLC). In this study, we have explored the possibility of exploiting inherent differences in KRAS-mutant cell metabolism for treatment. This study identified a greater dependency on folate metabolism pathways in KRAS mutant compared with KRAS wild-type NSCLC cell lines. Microarray gene expression and biologic pathway analysis identified higher expression of folate metabolism- and purine synthesis-related pathways in KRAS-mutant NSCLC cells compared with wild-type counterparts. Moreover, pathway analysis and knockdown studies suggest a role for MYC transcriptional activity in the expression of these pathways in KRAS-mutant NSCLC cells. Furthermore, KRAS knockdown and overexpression studies demonstrated the ability of KRAS to regulate expression of genes that comprise folate metabolism pathways. Proliferation studies demonstrated higher responsiveness to methotrexate, pemetrexed, and other antifolates in KRAS-mutant NSCLC cells. Surprisingly, KRAS gene expression is downregulated in KRAS wild-type and KRAS-mutant cells by antifolates, which may also contribute to higher efficacy of antifolates in KRAS-mutant NSCLC cells. In vivo analysis of multiple tumorgraft models in nude mice identified a KRAS-mutant tumor among the pemetrexed-responsive tumors and also demonstrated an association between expression of the folate pathway gene, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), and antifolate activity. Collectively, we identify altered regulation of folate metabolism in KRAS-mutant NSCLC cells that may account for higher antifolate activity in this subtype of NSCLC.

Background: VS-6766 is a unique small molecule inhibitor of both RAF and MEK. In contrast to several other MEK inhibitors available, VS-6766 blocks both MEK kinase activity and RAF phosphorylation of MEK. This sequential blockade mechanism enables VS-6766 to block MEK signaling more consistently without the compensatory and paradoxical activation of MEK that reduces the efficacy of other small molecule inhibitors of MEK and RAF. Defactinib (VS-6063), an orally active small molecule, is a potent adenosine 5'- triphosphate (ATP) competitive, reversible inhibitor of focal adhesion kinase (FAK). Defactinib has shown synergistic activity with BRAF and MEK inhibitors in both in vitro and in vivo preclinical solid tumor models. Specifically, in several human tumor cell lines with mutations in RAS or BRAF or wildtype RAS and BRAF, defactinib has shown synergy with MEK inhibitors or VS-6766. In mouse models of KRAS mutant ovarian cancer, BRAF-mutant melanoma or uveal melanoma, FAK inhibition has been shown to induce tumor regression when combined with RAF, MEK or RAF/MEK inhibitors, while the single agents have only induced tumor stasis. The combination of VS-6766 and defactinib is currently being evaluated in the Investigator Sponsored FRAME study. Preliminary efficacy results are available for a small number of subjects with KRAS mutated NSCLC treated with a combination of VS-6766 and defactinib. Of the 10 subjects with NSCLC, 1 subject with KRAS-G12V- mutated cancer achieved PR while 3/10 subjects received treatment for ≥24 weeks. In an updated analysis of response in 15 subjects with KRAS-mutant NSCLC, there were 2 PRs and 7 SDs (ORR: 13%). The two subjects with KRAS G12V- mutated NSCLC both achieved PR (ORR: 100%). Methods: This is an adaptive, two-part, Phase 2, multicenter, parallel cohort, randomized, open label study designed to evaluate the efficacy and safety of VS-6766 versus VS-6766 in combination with defactinib in subjects with recurrent NSCLC(NCT04620330). The study will be conducted in two parts. Part A will determine the optimal regimen, either VS-6766 monotherapy or VS-6766 in combination with defactinib based on confirmed overall response rate as assessed by independent radiology review. Part A will consist of three arms in KRAS mutated NSCLC Arm 1 monotherapy of VS-6766, Arm 2 the combination of VS-6766 and defactinib in KRAS G12V mutated and Arm 3 the combination in other KRAS mutated. Part B will determine the efficacy of the optimal regimen identified in Part A in KRAS mutated NSCLC. Subjects enrolled must have histologic or cytologic evidence of NSCLC, measurable disease according to RECIST V1.1 and known KRAS mutation. The study will enroll up to 377 subjects globally with 57 subjects (32 KRAS G12V & 25 KRAS other) in Part A and an additional 144 KRAS G12V and 176 KRAS other in Part B. This study is open for enrollment. Citation Format: D. Ross Camidge, Jonathan Pachter, Andrew Koustenis, Gloria Patrick, David R. Spigel. A phase 2 study of VS-6766 (dual RAF/MEK inhibitor) RAMP 202, as a single agent and in combination with defactinib (FAK inhibitor) in recurrent KRAS-mutant (KRAS-MT) non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P048.

IntroductionOncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. We have previously shown that NSCLC cells harbouring KRAS mutations are more sensitive than their wild-type counterparts to inhibition of KRAS downstream effectors MEK and RAF and to treatment with IGF1R inhibitors.Material and methodsIn order to identify complementary targets for the improvement of IGF1R and/or MEK targeting therapies, we performed a whole-genome shRNA screen with KRAS-mutant NSCLC cells. Validation was performed using shRNAs and small molecule inhibitors in a panel of NSCLC cell lines. In vivo efficacy of drug combinations was measured in mouse models of Kras-induced NSCLC using CT scanning.Results and discussionsThe list of sensitizers to IGF1R inhibitors included several genes encoding components of the mTOR pathway. Viability assays in a panel of lung cancer cell lines confirmed that combining IGF1R inhibitors with mTOR inhibitors, both rapalogs and kinase inhibitors, resulted in a synergistic anti-proliferative effect in KRAS-mutant NSCLC cells. Mechanistic investigations demonstrated that IGF1R inhibitors blocked reactivation of the PI3K pathway induced by mTOR inhibition, resulting in a robust suppression of PI3K and mTORC1 signalling. Addition of a MEK inhibitor to the combination produced a more profound and durable suppression of cell proliferation and a stronger induction of apoptosis by inhibiting the main downstream pathways controlled by KRAS. Notably, the inhibition of these signalling pathways was stronger in KRAS-mutant cells than in wild-type cells. In order to achieve strong downstream pathway inhibition even more specifically in KRAS-mutant cells, we used the KRAS-G12C mutant inhibitor, ARS-1620. Interestingly, addition of mTOR and IGF1R inhibitors vastly increases effectiveness of the KRAS-G12C inhibitor.Finally, we validated the drug combinations in mouse models of KRAS-induced NSCLC. Results showed that combined mTOR, IGF1R and MEK inhibition produced a marked tumour regression in a NSCLC mouse model driven by mutant Kras and p53 loss-of-function and also in urethane-induced lung tumours.ConclusionWe have demonstrated that a profound inhibition of the main pathways downstream KRAS is needed to achieve a durable suppression of cell viability in NSCLC cells harbouring KRAS mutations. These findings suggest potential novel therapeutic strategies for KRAS mutant NSCLC tumours.

An integrative pharmacogenomics analysis identifies therapeutic targets in KRAS-mutant lung cancer.

This study aimed to confirm whether Kirsten rat sarcoma viral oncogene (KRAS) mutations affect the therapeutic efficacy of non-small cell lung cancer (NSCLC) and, if so, to explore what the possible mechanisms might be. We retrospectively analyzed the efficacy of immunochemotherapy in KRAS-mutant NSCLC patients compared to driver-negative patients. Online data platforms were used to find immunotherapy cases, and survival analysis compared treatments' efficacy. Cytotoxicity assays measured chemosensitivity in KRAS-mutant versus wild-type NSCLC to drugs like paclitaxel, carboplatin, and pemetrexed. Bioinformatics confirmed the KRAS-SLC7A11 link and cell experiments tested SLC7A11's role in chemoresistance. Animal studies verified the antitumor effects of SLC7A11 inhibitors with chemotherapy. Patients with KRAS-mutated NSCLC have a shorter therapeutic effectiveness duration with immunochemotherapy than patients with driver gene-negative status. The efficacy of immunotherapy alone is similar between the two groups. The KRAS mutation can enhance chemoresistance by upregulating SLC7A11, and inhibiting SLC7A11 can increase the sensitivity of KRAS-mutated NSCLC to chemotherapy. This study suggests that KRAS-mutant NSCLC can enhance its acquired chemoresistance by overexpressing SLC7A11, leading to poorer therapeutic outcomes. Targeting the KRAS-SLC7A11 axis could increase sensitivity to chemotherapeutic drugs, providing theoretical support for future treatment directions.

Oncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. Thus, new therapeutic strategies are needed to treat these tumors. We have previously shown that NSCLC cells harboring KRAS mutations are more sensitive to inhibition of KRAS downstream effectors MEK and RAF and to treatment with IGF1R inhibitors than their wild-type counterparts. In order to identify complementary targets for the improvement of IGF1R and/or MEK targeting therapies, we performed a whole-genome shRNA screen in KRAS-mutant NSCLC cells. Interestingly, the list of sensitizers to IGF1R inhibitors included several genes encoding components of the mTOR pathway. Viability assays in a panel of lung cancer cell lines confirmed that combinations of IGF1R inhibitors with mTOR inhibitors, both rapalogs and kinase inhibitors, resulted in a synergistic anti-proliferative effect in KRAS-mutant NSCLC cells. Mechanistic investigations demonstrated some differences between the effects of rapalogs and mTOR kinase inhibitors although, in both cases, mTOR inhibitors increased IGF1R and AKT phosphorylation. Combination with IGF1R inhibitors blocked the reactivation of the PI3K pathway resulting in a robust suppression of PI3K and mTORC1 signaling. Notably, the inhibition of these signaling pathways was stronger in KRAS-mutant cells than in wild-type cells. Addition of a MEK inhibitor to the combination produced a more profound and durable suppression of cell proliferation and a stronger induction of apoptosis. Finally, we validated the different drug combinations in mouse models of KRAS-induced NSCLC. Results showed that the three-drug combination produced a marked tumor regression in a NSCLC mouse model driven by mutant Kras and p53 loss-of-function and also in urethane-induced lung tumors. These findings suggest potential novel therapeutic strategies for NSCLC tumors harboring KRAS mutations. Citation Format: Miriam Molina-Arcas, Christopher Moore, Sareena Rana, Febe van Maldegem, Stuart Horswell, David Hancock, Julian Downward. Identification of new combination therapies for lung tumors harboring KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2987.

KRAS-mutant non-small cell lung cancer (NSCLC) is a major lung cancer subtype that leads to many cancer-related deaths worldwide. Although numerous studies on KRAS-mutant type NSCLC have been conducted, new oncogenic or tumor suppressive genes need to be detected because a large proportion of NSCLC patients does not respond to currently used therapeutics. Here, we show the tumor-promoting function of a cell cycle-related protein, PIERCE1, in KRAS-mutant NSCLC. Mechanistically, PIERCE1 depletion inhibits cell growth and AKT phosphorylation (pAKT) at S473, which is particularly observed in KRAS-mutant lung cancers. Analyses of AKT-related genes using microarray, immunoblotting, and real-time quantitative PCR indicated that PIERCE1 negatively regulates the gene expression of the AKT suppressor, TRIB3, through the CHOP pathway, which is a key regulatory pathway for TRIB3 expression. Similarly, in vivo analyses of PIERCE1 depletion in the KRAS mutation-related lung cancer mouse models revealed the suppressive effect of PIERCE1 knockout in urethane- and KRASG12D-induced lung tumorigenesis with decreased pAKT levels observed in the tumors. Tissue microarrays of human lung cancers indicated the expression of PIERCE1 in 83% of lung cancers and its correlation with pAKT expression. Thus, we illustrate how PIERCE1 depletion may serve as a therapeutic strategy against KRAS-mutant NSCLC and propose the clinical benefit of PIERCE1.

The recently approved KRASG12C mutation-specific inhibitors sotorasib and adagrasib (KRASG12C-I) represent a promising therapy for KRASG12C-driven non-small cell lung cancer (NSCLC). However, many eligible patients do not benefit due to intrinsic or acquired drug resistance. Tissue factor (TF) is overexpressed in KRAS-mutated (KRASmut) NSCLC and is the target of the FDA-approved ADC Tivdak. Here, we employed HuSC1-39, the parent antibody of a clinical stage TF-ADC (NCT04843709), to investigate the role of TF in KRASmut NSCLC. We found that patients with TF-overexpression had poor survival, elevated P-ERK/P-AKT activity levels and low immune effector cell infiltration in the tumor. In a panel of KRASG12C cell lines, KRASG12C-I response correlated with suppression of TF mRNA, which was not observed in resistant cells. In the drug resistant cells, TF-overexpression relied on an mTORC2-mediated and proteasome-dependent pathway. Combination treatment of HuSC1-39 or mTORC1/2 inhibitor MTI-31 with KRASG12C-I each produced synergistic antitumor efficacy in cell culture and in an orthotopic lung tumor model. TF-depletion in the resistant cells diminished epithelial mesenchymal transition, reduced tumor growth and greatly sensitized KRASG12C-I response. Moreover, employing immunohistochemistry and coculture studies, we demonstrated that HuSC1-39 or MTI-31 reset the tumor microenvironment and restore KRASG12C-I sensitivity by reshaping an M1-like macrophage profile with greatly enhanced phagocytic capacity toward tumor cell killing. Thus, we have identified the TF/mTORC2 axis as a critical new mechanism for triggering immunosuppression and KRASG12C-I resistance. We propose that targeting this axis with HuSC1-39 or MTI-31 will improve KRASG12C-I response in KRAS-driven NSCLC.

Efficacy of Immune Checkpoint Inhibitors in KRAS-Mutant Non-Small Cell Lung Cancer (NSCLC)