KRAS Mutant Cell Lines Research Articles

Autophagy inhibitors increase the susceptibility of KRAS-mutant human colorectal cancer cells to a combined treatment of 2-deoxy-D-glucose and lovastatin.

RAS-driven colorectal cancer relies on glucose metabolism to support uncontrolled growth. However, monotherapy with glycolysis inhibitors like 2-deoxy-D-glucose causes limited effectiveness. Recent studies suggest that anti-tumor effects of glycolysis inhibition could be improved by combination treatment with inhibitors of oxidative phosphorylation. In this study we investigated the effect of a combination of 2-deoxy-D-glucose with lovastatin (a known inhibitor of mevalonate pathway and oxidative phosphorylation) on growth of KRAS-mutant human colorectal cancer cell lines HCT116 and LoVo. A combination of lovastatin (>3.75 μM) and 2-deoxy-D-glucose (>1.25 mM) synergistically reduced cell viability, arrested cells in the G2/M phase, and induced apoptosis. The combined treatment also reduced cellular oxygen consumption and extracellular acidification rate, resulting in decreased production of ATP and lower steady-state ATP levels. Energy depletion markedly activated AMPK, inhibited mTOR and RAS signaling pathways, eventually inducing autophagy, the cellular pro-survival process under metabolic stress, whereas inhibition of autophagy by chloroquine (6.25 μM) enhanced the cytotoxic effect of the combination of lovastatin and 2-deoxy-D-glucose. These in vitro experiment results were reproduced in a nude mouse xenograft model of HCT116 cells. Our findings suggest that concurrently targeting glycolysis, oxidative phosphorylation, and autophagy may be a promising regimen for the management of RAS-driven colorectal cancers.

KHDRBS3 promotes multi-drug resistance and anchorage-independent growth in colorectal cancer.

5‐Fluorouracil (5‐FU) is one of the most frequently used pharmacological agents in the treatment of colorectal cancer (CRC). Resistance to chemotherapy is a major cause of treatment failure of CRC, and it is a well known fact that cancer stem cells play a significant role in the acquisition of drug resistance. In this study, we focused on the KHDRBS3 gene that encodes KH RNA Binding Domain Containing, Signal Transduction Associated 3. We first clarified the relationship between KHDRBS3 and 5‐FU resistance. We then observed higher expression levels of KHDRBS3 in KRAS‐mutant organoids and cell lines in comparison with KRAS wild‐type organoids and cell lines. Immunohistochemical analysis using CRC cases revealed that the prognosis of KHDRBS3‐positive patients was significantly worse compared with that of KHDRBS3‐negative patients. Univariate and multivariate Cox proportional hazards analyses showed that KHDRBS3 was an independent prognostic factor in patients with CRC. We determined that KHDRBS3 might play a crucial role in the acquisition of stem cell properties, such as drug resistance and spheroid/organoid formation, by regulating CD44 variant expression and the Wnt signaling pathway. In an immunodeficient mouse model, KHDRBS3‐positive cells showed efficient tumor formation and formed metastatic lesions in the lungs. These results indicated that KHDRBS3 plays a crucial role in drug resistance and anchorage‐independent growth by maintaining stem cell‐like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients.

AZD0364 Is a Potent and Selective ERK1/2 Inhibitor That Enhances Antitumor Activity in KRAS-Mutant Tumor Models when Combined with the MEK Inhibitor, Selumetinib.

The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

KRAS G12V mutation upregulates PD-L1 expression via TGF-β/EMT signaling pathway in human non-small-cell lung cancer.

Although clinical data suggest remarkable promise for targeting programmed cell death protein-1 (PD-1) and ligand (PD-L1) signaling in non-small-cell lung cancer (NSCLC), it is still largely undetermined which subtype of patients will be responsive to checkpoint blockade. In the present study, we explored whether PD-L1 was regulated by mutant Kirsten rat sarcoma viral oncogene homolog (KRAS), which is frequently mutated in NSCLC and results in poor prognosis and low survival rates. We verified that PD-L1 levels were dramatically increased in KRAS mutant cell lines, particularly in NCI-H441 cells with KRAS G12V mutation. Overexpression of KRAS G12V remarkably elevated PD-L1 messenger RNA and protein levels, while suppression of KRAS G12V led to decreased PD-L1 levels in NCI-H441 cells. Consistently, higher levels of PD-L1 were observed in KRAS-mutated tissues as well as tumor tissues-derived CD4+ and CD8+ T cells using a tumor xenograft in B-NDG mice. Mechanically, both in vitro and in vivo assays found that KRAS G12V upregulated PD-L1 via regulating the progression of epithelial-to-mesenchymal transition (EMT). Moreover, pembrolizumab activated the antitumor activity and decreased tumor growth with KRAS G12V mutated NSCLC. This study demonstrates that KRAS G12V mutation could induce PD-L1 expression and promote immune escape via transforming growth factor-β/EMT signaling pathway in KRAS-mutant NSCLC, providing a potential therapeutic approach for NSCLC harboring KRAS mutations.

Whole Transcriptome Sequencing Analysis of Cancer Stem/Progenitor Cells Obtained from Mouse Lung Adenocarcinomas.

Interrogation and characterization of lung cancer stem cells (CSCs) that are implicated in lung oncogenesis is crucial for our understanding of inter- and intra-tumor heterogeneity and the aggressive nature of the disease, including tumor resistance and relapse in response to conventional therapy. Here, we describe an in vitro surrogate model, namely the "sphere-forming assay," for the derivation, enrichment, and propagation of lung stem/progenitor cells with CSC properties, including self-renewal, tumor initiation capacity and propagation, and differentiation into cells of the tumor bulk, from a murine Kras-mutant lung adenocarcinoma cell line. Self-renewing cancer stem/progenitor cells, in the form of 3D in vitro spheres, can be phenotypically interrogated using downstream techniques such as gene expression analysis (e.g., whole transcriptome sequencing and quantitative real time PCR), which is the focus of this chapter.

KRAS-regulated glutamine metabolism requires UCP2-mediated aspartate transport to support pancreatic cancer growth.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.

Co-Targeting BET Bromodomain Proteins and Aberrant Signaling in AML

AML remains a therapeutic challenge with a high mortality rate, underscoring the need for new biologically based therapies. Somatic alterations that deregulate epigenetic programs and signal transduction pathways frequently coexist in AML. While the former class of alterations is hypothesized to promote a chromatin state that is permissive for AML development and essential for leukemia maintenance, experimental data also suggest that signaling mutations play a central role in driving leukemic growth. Thus, simultaneously targeting the abnormal epigenetic landscape and aberrant signaling is a rational therapeutic approach. We previously used the MEK inhibitor PD0325901 (PD901) to target the Raf/MEK/ERK (MAPK) effector pathway in primary murine AMLs in vivo. Here we report substantial preclinical efficacy of the BET inhibitor PLX51107 in several independent primary AMLs that was further enhanced by PD901. We first exposed five NRAS or KRAS mutant human AML cell lines to varying concentrations of PLX51107 and PD901. These studies revealed potent synergy based on cell proliferation and apoptosis readouts (Figure 1A). We next performed RNA-seq on the NRAS-mutant OCI-AML3 cell line treated with PD901, PLX51107, or the combination. Remarkably, PLX51107 + PD901 cooperated to potently downregulate MYC and its transcriptional targets (Figure 1B). Additionally, AML cells increased the expression of p53 transcriptional targets in response to PLX51107 and CRISPRi-mediated knock-down of TP53 expression abrogated the synergistic effects of PLX51107 + PD901 (Figure 1C). We previously performed retroviral insertional mutagenesis in NrasG12D mice to generate genetically diverse panels of transplantable, primary AMLs. To follow-up on our promising in vitro data, we transplanted 5 of these leukemias into cohorts of recipient mice and treated them with PLX51107 ± PD901. PLX51107 (10 mg/kg/day) showed impressive efficacy that was further enhanced by a modest dose of PD901 (1.5 mg/kg given 4 days per week; Figure 1D). To begin characterizing mechanisms of resistance, we evaluated matched pairs of drug-sensitive ("parental") and relapsed leukemias that emerged during drug treatment. We selected relapsed leukemias that exhibited clonal evolution based on distinct novel retroviral integrations for in depth evaluation. Re-transplanting these AMLs into secondary recipients and re-treating them with PLX51107 ± PD901 confirmed intrinsic drug resistance, which was further validated ex vivo in dose response methylcellulose assays (Figure 1D). Remarkably, several of these resistant leukemias down-regulated basal Myc protein expression, which supports the idea that their transcriptional networks are "rewired" (Figure 1E). We next compared basal and dynamic transcriptional profiles in parental and resistant leukemias by exposing them to PLX51107 ex vivo and then performing RNA-seq analysis. Intriguingly, PLX51107 treatment of parental AMLs resulted in transcriptional changes consistent with myeloid maturation in a dose dependent manner (Figure 1E), suggesting that BET inhibition induces differentiation in these leukemias. By contrast, paired resistant AMLs are relatively "immature" compared to their parental counterparts (Figure 1E), which supports this idea that in vivo treatment with PLX51107 selects for the outgrowth of clones at an earlier progenitor state. These resistant AMLs also upregulate Myc transcriptional targets and unexpectedly downregulate Ras transcriptional targets (Figure 1E). We corroborated these findings by analyzing large pediatric AML transcriptome datasets generated as part of the NCI TARGET initiative (Figure 1F). Taken together, our data support the hypothesis that BET inhibitor and MEK inhibitor combination may be a particularly effective therapeutic approach in AML. Additional preclinical testing in patient derived xenograft models is underway. Figure 1 Disclosures Burgess: Bristol Myers Squibb: Current Employment. Severson:Plexxikon Inc.: Current Employment. Powell:Plexxikon Inc.: Current Employment. Bollag:Plexxikon Inc.: Current Employment.

Activity of Birinapant, a SMAC Mimetic Compound, Alone or in Combination in NSCLCs With Different Mutations.

Liver kinase B1 (LKB1/STK11) is the second tumor suppressor gene most frequently mutated in non-small-cell lung cancer (NSCLC) and its activity is impaired in about half KRAS-mutated NSCLCs. Nowadays, no effective therapies are available for patients having these mutations. To highlight new vulnerabilities of this subgroup of tumors exploitable to design specific therapies we screened an US FDA-approved drug library using an isogenic system of wild-type (WT) or deleted LKB1. Among eight hit compounds, Birinapant, an inhibitor of the Inhibitor of Apoptosis Proteins (IAPs), was the most active compound in LKB1-deleted clone only compared to its LKB1 WT counterpart. We validated the Birinapant cells response and its mechanism of action to be dependent on LKB1 deletion. Indeed, we demonstrated the ability of this compound to induce apoptosis, through activation of caspases in the LKB1-deleted clone only. Expanding our results, we found that the presence of KRAS mutations could mediate Birinapant resistance in a panel of NSCLC cell lines. The combination of Birinapant with Ralimetinib, inhibitor of p38α, restores the sensitivity of LKB1- and KRAS-mutated cell lines to the IAP inhibitor Birinapant. Our study shows how the use of Birinapant could be a viable therapeutic option for patients with LKB1-mutated NSCLCs. In addition, combination of Birinapant and a KRAS pathway inhibitor, as Ralimetinib, could be useful for patients with LKB1 and KRAS-mutated NSCLC.

PLCγ1 suppression promotes the adaptation of KRAS-mutant lung adenocarcinomas to hypoxia.

Mutant KRAS modulates the metabolic plasticity of cancer cells conferring growth advantage during hypoxia, but the molecular underpinnings are largely unknown. Using a lipidomic screen, we found that PLCγ1 is suppressed during hypoxia in KRAS-mutant human lung adenocarcinoma cancer cell lines. Suppression of PLCγ1 in hypoxia promotes a less oxidative cancer cell metabolism, reduces the formation of mitochondrial reactive oxygen species and switches tumor bioenergetics towards glycolysis by impairing Ca2+ entry into the mitochondria. This event prevents lipid peroxidation, antagonizes apoptosis and increases cancer cell proliferation. Accordingly, loss-of-function of Plcγ1 in a mouse model of KrasG12D-driven lung adenocarcinoma increased the expression of glycolytic genes, boosted tumor growth and reduced survival. In patients with mutant KRAS lung adenocarcinomas, low PLCγ1 expression correlates with increased expression of hypoxia markers and predicts poor patient survival. Thus, our work reveals a mechanism of cancer cell adaptation to hypoxia with potential therapeutic value.

Targeting antioxidant enzymes enhances the therapeutic efficacy of the BCL-XL inhibitor ABT-263 in KRAS-mutant colorectal cancers

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-XL, by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-XL targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations.

Chemotherapeutic effects of MEK kinase inhibitor and BRAF kinase inhibitor on KRAS-mutated human colon cancer cell lines with different microsatellite instability

We analyzed responsiveness of KRAS-mutated CRC cell lines with distinctive MSI status against mitogen-activated protein kinase (MEK) inhibitor (selumetinib; AZD) and/or B-raf proto-oncogene (BRAF) kinase inhibitor (vemurafenib; PLX). The viability of MSI-high (MSI-H) KRAS-mutated LS174T cells treated with AZD or PLX was 24.5 ± 0.9% or 71.4 ± 3.6%, respectively, and the viability of microsatellite stable (MSS) KRAS-mutated SW480 cells for AZD or PLX was 57.4 ± 3.1% or 43.1 ± 1.8%, respectively. These observations imply that the therapeutic efficacy of MEK kinase inhibitors or BRAF kinase inhibitors against KRAS-mutated colon cancer cells may differ between MSI-H and MSS. However, a combination of both inhibitors synergistically inhibits the proliferation of KRAS-mutated colon cancer cells regardless of MSI status. The underlying synergistic cytotoxic efficacy of AZD/PLX combination on KRAS-mutated colon cancer cells with different MSI status was further substantiated by markedly decreased phosphorylation of ERK in both LS74T and SW480 cell lines upon AZD and PLX treatment. Based on these collective data, we propose that MSI status should be considered when MEK kinase inhibitor or BRAF kinase inhibitor is treated for KRAS-mutated colon cancer, and that combination of both inhibitors synergistically inhibit proliferation of KRAS-mutated colon cancer cells independent of MSI status.

Abstract B06: Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses Kras-mutated non-small cell lung cancer

Abstract Activating KRAS mutation is the most common driver mutation in the initiation and promotion of non-small cell lung cancer (NSCLC). Direct inhibition of the enzymatic activity of mutated Kras has been proved unsuccessful. Deregulation of PI3K-Akt-mTOR signaling, one of the most important downstream effectors of Kras, plays important roles in tumorigenesis and drug resistance in NSCLC. In this study, we found that a novel PI3Kα-specific inhibitor, CYH33, possessed variable activity against a panel of Kras-mutated NSCLC cell lines. Though CYH33 blocked Akt phosphorylation in all tested cells, phosphorylated Rb decreased in CYH33-sensitive but not resistant cells, which was consistent with G1 phase arrest in CYH33-sensitive cells. Consequently, combination of a CDK4/6 inhibitor PD0332991 with CYH33 displayed synergistic activity against the proliferation of both CYH33-sensitive and -resistant cells, which was accompanied with enhanced cell population in G1 phase compared to single-agent treatment. Moreover, downregulation of cyclin D1 with siRNA sensitized A549 and H23 cells to CYH33. Reciprocally, CYH33 abrogated PD0332991-induced upregulation of phosphorylated Akt and cyclin D1 in A549 cells. Cotreatment of these two drugs demonstrated synergistic activity against A549 and H23 xenografts with enhanced inhibition on the phosphorylation of Akt and Rb compared to administration of CYH33 or PD03332991 alone. Thus, simultaneous inhibition of PI3Kα and CDK4/6 displayed synergistic activity against Kras-mutated NSCLC and provided a mechanistic rationale for a combination approach using PI3Kα inhibitor and CDK4/6 inhibitor CYH33 for the therapy of Kras-mutated NSCLC. Citation Format: Yu-xiang Wang, Xian Li, Xue-ling Liu, Bo-bo Wang, Yi Chen, Chun-hao Yang, Jian Ding, Ling-hua Meng. Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses Kras-mutated non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B06.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

In-Vitro and In-Silico Evaluations of Heterocyclic-Containing Diarylpentanoids as Bcl-2 Inhibitors Against LoVo Colorectal Cancer Cells.

In the present study, we investigated the in-vitro anti-cancer potential of six diarylpentanoids against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, SW620, LoVo, HT29, NCI-H508, RKO, and LS411N cells. Structure–activity relationship study suggested that the insertions of tetrahydro-4H-thiopyran-4-one and brominated phenyl moieties are essential for better cytotoxicity. Among the evaluated analogs, 2e has been identified as the lead compound due to its low IC50 values of approximately 1 µM across all cancer cell lines and high chemotherapeutic index of 7.1. Anti-proliferative studies on LoVo cells showed that 2e could inhibit cell proliferation and colony formations by inducing G2/M cell cycle arrest. Subsequent cell apoptosis assay confirmed that 2e is a Bcl-2 inhibitor that could induce intrinsic cell apoptosis by creating a cellular redox imbalance through its direct inhibition on the Bcl-2 protein. Further molecular docking studies revealed that the bromophenyl moieties of 2e could interact with the Bcl-2 surface pocket through hydrophobic interaction, while the tetrahydro-4H-thiopyran-4-one fragment could form additional Pi-sulfur and Pi-alkyl interactions in the same binding site. In all, the present results suggest that 2e could be a potent lead that deserves further modification and investigation in the development of a new Bcl-2 inhibitor.

Abstract 4384: Quantitative assessment of drug combination effects in NSCLC cell lines through network-based analysis of functional protein activity

Abstract Non-small cell lung cancer (NSCLC) is the most prevalent type of lung cancer, which is the leading cause of cancer death worldwide. The KRAS oncogene is one of the most common driver mutations in NSCLC patients but, despite that, there are no approved targeted therapies for tumors harboring this mutation. The MEK inhibitor Selumetinib (SE) turned out to be an ineffective drug against KRAS mutated lung cancer. Since combination therapy has shown to be beneficial in many tumors, we wanted to investigate the combination of MEK inhibition with other targeted therapies. Thus, we measured the proteomic response of KRAS mutant (MUT) and wild-type (WT) NSCLC cell lines (CLs) to the combination of SE with Everolimus (EV), an mTOR inhibitor, and Tamoxifen (TA), an Estrogen inhibitor. The expression level of 183 proteins was measured through Reverse Phase Protein Array (RPPA) in eight NSCLC CLs treated with SE, SE plus EV and SE plus TA at 6 time points: 5 minutes (min), 30 min, 1, 2, 6 and 24 hours (h). As control, measures were taken in baseline CLs and in CLs with only dimethyl sulfoxide. To study the drug combination effects, we applied a computational workflow centered on two algorithms initially developed for gene expression data. We analyzed the CLs with the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) to reconstruct a context specific signaling network of 2635 interactions between 73 kinases and 6 phosphatases and their substrates. Then, we analyzed the obtained network with the Virtual Inference of Protein activity by Enriched Regulon analysis (VIPER) algorithm to assess the mechanism of action of the drug compounds and how they dysregulate the activity of kinases and phosphatases over time. With respect to SE alone and apart from the drug targets, VIPER predicted the following proteins as highly inhibited over time across all CLs : 1) Aurora and LKB1 in SE+EV, 2) AMPK, c-RAF and ATR in SE+TA, 3) mTOR, p90, p70S6K, c-MET and GSK-3 in both combinations. On the other hand, Akt, ETK, Axl and FAK are all activated in SE+EV as well as PP2A, SGK1 and IGF1R in SE+TA, after 1h from the treatment. By running Gene Set Enrichment Analysis on the VIPER output against the primary drug target pathways, we found that mTOR signaling pathway exhibits a quite different evolution between KRAS MUT and WT CLs. On the other hand, other pathways revealed mechanisms of response that are CL specific. For instance, the main MEK pathways in H1734 CL (KRAS MUT) treated with SE+TA are all insensitive to the drug compounds. In conclusion, this computational approach successfully predicted protein-protein interactions and elucidated both proteomic mechanisms of drug combinations and CL specific dependencies. It allows to develop effective predictive and quantitative models reproducing each CL behavior, in order to realize the fullest potential of a targeted therapeutic approach. Citation Format: Chiara Antonini, George Rosenberger, Fortunato Bianconi, Lorenzo Tomassoni, Vienna Ludovini, Sara Baglivo, Sara Calandrini, Elisa Baldelli, Mariaelena Pierobon, Emanuel F. Petricoin, Andrea Califano. Quantitative assessment of drug combination effects in NSCLC cell lines through network-based analysis of functional protein activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4384.

Abstract 1957: Repotrectinib increases effectiveness of MEK inhibitor trametinib in KRAS mutant cancer models via simultaneous SRC/FAK/JAK2 inhibition

Abstract Kirsten Rat Sarcoma Viral Oncogene homolog (KRAS) is the most frequently mutated oncogene in a broad spectrum of human cancers, including ~30% of non-small cell lung cancer (NSCLC), ~45% of colorectal cancer (CRC) and ~75% of pancreatic cancer. The MEK inhibitor trametinib, which targets the primary downstream signaling pathway activated by mutant KRAS, has shown limited activity when used as a single agent in patients with previously treated KRAS-mutant positive NSCLC. Compensatory upregulation of the PI3K/AKT survival signaling and intrinsic or treatment induced epithelial-to-mesenchymal transition (EMT) are among resistance mechanisms of MEK inhibition in KRAS mutant cancers. SRC/FAK signaling modulates the PI3K/AKT survival pathway and is involved in the integrin mediated EMT signaling. In addition, MEK inhibition has been reported to activate the JAK2/STAT3 pathway to evade therapy. Therefore, the combination of a MEK inhibitor with agents that simultaneously inhibit SRC, FAK and JAK2 may represent a promising therapeutic approach to effectively target KRAS mutant cancers. Repotrectinib, a next generation ROS1/TRK/ALK investigational drug, also inhibits SRC/FAK/JAK2 at therapeutically relevant concentrations. Here, we present synergistic effects of repotrectinib in combination with trametinib in KRAS mutant cancer models. Increased anti-proliferative activity was demonstrated by the combination across panels of KRAS mutant NSCLC and CRC cell lines that harbor a spectrum of KRAS mutations. Additionally, the combination of trametinib and repotrectinib in KRAS mutant cell lines caused an enhanced induction of apoptosis in comparison with trametinib or repotrectinib alone. Trametinib induced a rebound in phospho-AKT (pAKT) levels in a subset of KRAS mutant cells which was substantially suppressed by the combination with repotrectinib. In contrast, suppression of the trametinib-induced pAKT rebound was not achieved by combinations of trametinib with dasatinib (SRCi), ruxolitinib (JAK1/2i), or defactinib (FAKi), suggesting that concomitant SRC/FAK/JAK2 inhibition is potentially necessary. Combination of trametinib and repotrectinib in KRAS mutant murine xenograft studies resulted in suppression of SRC/FAK/STAT3/EGFR/ERK phosphorylation and the enhancement of tumor apoptosis. Importantly, the combination of repotrectinib and trametinib demonstrated increased in vivo anti-tumor effect compared to trametinib alone. Our data suggest that the combination of repotrectinib with trametinib may sustainably suppress the mutant KRAS network signaling to achieve more potent and durable anti-tumor efficacy, and that further clinical investigation of the combination treatment of a MEK inhibitor with repotrectinib in patients with KRAS mutant cancers is warranted. Citation Format: Brion Murray, Wei Deng, Dayong Zhai, Laura Rodon, Nathan Lee, J. Jean Cui. Repotrectinib increases effectiveness of MEK inhibitor trametinib in KRAS mutant cancer models via simultaneous SRC/FAK/JAK2 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1957.

Abstract 1825: Tumor-targeted knockdown of KRAS mutants with novel Centyrin:siRNA conjugates

Abstract KRAS has been the quintessential undruggable oncogene for many years. Although there have been recently described small molecule inhibitors of KRAS, these molecules only target a specific mutant form, KRAS G12C. The KRAS G12C mutation represents just 12% of the total number of KRAS mutants across many different tumor types including CRC, NSCLC, and PDAC. We have taken a novel approach to decrease all KRAS mRNA and protein mutants using Centyrin:siRNA drug conjugates which should have applicability across a broader group of patients. Centyrins are a new class of small, stable and highly soluble proteins engineered to bind antigens with high affinity. Centyrins facilitate internalization of macromolecules, including siRNA, into cells and enable trafficking through endosomes and into the cytoplasm. Centyrins are engineered by consensus design from human tenascin C with each domain having a MW of approximately 10 kilodaltons. We have rationally designed a bispecific Centyrin conjugate which targets EpCAM and CD71 to deliver an optimized, chemically modified pan-KRAS siRNA into epithelial derived tumors. EpCAM and CD71 (the transferrin receptor) were selected due to their higher levels of expression on tumors versus normal tissue and the targeting of these receptors with a bispecific molecule enhances both potency of siRNA delivery and selectivity to tumor cells. In addition, Centyrin:siRNA conjugate candidates contain a domain to extend in vivo exposure. The designed Centyrin:siRNA drug conjugates exhibit potencies for knockdown of KRAS mRNA and protein at nM concentrations across various KRAS mutant tumor cell lines, resulting in potent inhibition of proliferation. We report activity on tumor cell lines harboring all of the most common KRAS mutants including G12D (HPAF-II), G12V (SW620), G12S (A549), G12C (H358, MIA PaCa-2), G13D (H1944), and Q61H (H460). This activity is specific for cells expressing EpCAM and CD71 and synergy of the bispecific versus the monospecific Centyrin has been demonstrated. The effect of the Centyrin:siRNA conjugate is specific for KRAS with no effect on the closely related homologs NRAS or HRAS. Pulse washout experiments suggest that in vitro treatment of cells for only 6-24 hours resulted in maximal knockdown of KRAS mRNA up to at least 72 hours. Preliminary pharmacokinetic studies have demonstrated good exposure (~100-400nM) of the siRNA in tumors in a xenograft model. Current efforts are aimed at demonstration of in vivo efficacy in multiple xenograft models with optimization of dose and scheduling. These novel Centyrin:siRNA conjugates have great potential to treat the many patients with KRAS mutations. Citation Format: Russell C. Addis, Robert Kolakowski, Swapnil Kulkarni, Joshua Gorsky, Rebecca Meyer, Yao Xin, Evana Mortezavi, Karyn T. O'Neil, Steven G. Nadler. Tumor-targeted knockdown of KRAS mutants with novel Centyrin:siRNA conjugates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1825.

Abstract 5246: Disulfiram anticancer metabolite, dithiocarb-copper complex, demonstrates anti-neoplastic activity against hypermutated colorectal cancers

Abstract Introduction: Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. It was responsible for about 51,000 deaths during 2019. Surgical resection and systemic chemotherapy are standard approaches to treat both localized and metastatic CRC (mCRC). However, the effect of the chemotherapy depends on the characteristics of the individual tumor, for instance CRC subtypes with a microsatellite instability (MSI) tend to have better prognosis and response to both chemotherapy and immune check-point inhibition. Recently, Disulfiram's anti-cancer activity was explored and its metabolite ditiocarb-copper complex (CuET) was found to have high anti-cancer activity. The mechanism of action of CuET was proposed to act through p97 segregase adaptor NPL4 and activation of endoplasmic reticulum stress leading to preferential apoptosis of cancer cells. Since altered protein degradation pathway is targeted by CuET, this compound might display high efficacy in highly mutated colorectal cancers. Methods: To assess CuET anti-cancer activity in vitro, we performed survival curve assays and colony-formation assays in both murine hypermutated colorectal cancer cell line MC-38 as well as in human KRAS mutant and MSI HCT116 cell line. We also evaluated anti-metastatic effect of CuET on both cell lines through migration-invasion assays in Boyden chamber setting. We assessed in vivo efficacy of systemic treatment with CuET alone or in combination with irinotecan, against MC-38 model in immunocompetent C57BL/6 mice. We characterized tumor growth, mice survival and tumor histopathology. Results: Our results demonstrate that CuET displays cytotoxicity in MC-38 and HCT116 cells at very low doses with respective IC50 of 46nM and 88nM. CuET also significantly reduces the ability of both cell lines to grow and form colonies under sustained treatment. Additionally, CuET is effective in significantly reducing cancer cell migration and invasion through porous membrane in vitro. Furthermore, systemic treatment with CuET significantly reduced tumor growth in mice and prolonged survival when compared to controls. Conclusion: CuET demonstrates cytotoxic activity against hypermutated colorectal cancer cells both in vitro and in vivo and should be further investigated in clinical trials. Citation Format: Daciana Catalina Dumut, Juan B. De Sanctis, Martin Mistrik, Zdenek Skrott, Petr Dzubak, Jiri Bartek, Marian Hajduch, Danuta Radzioch. Disulfiram anticancer metabolite, dithiocarb-copper complex, demonstrates anti-neoplastic activity against hypermutated colorectal cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5246.

Abstract 5710: Autophagy inhibition sensitizes Liver Kinase B1 (LKB1) - deficient Kras-driven lung tumors to MEK inhibitor Trametinib

Abstract Tumor suppressor liver kinase B1 (LKB1) activates 5'-adenosine monophosphate protein kinase (AMPK) and plays an essential role in maintaining energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non-small cell lung cancer (NSCLC). Compared to p53 mutation, co-mutations in LKB1 with KRAS cause aggressive tumor growth, metastases and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1-deficient Kras-driven NSCLC treatment is urgently needed. Our previous work demonstrates that autophagy inhibition is synthetically lethal in KrasG12D/+;Lkb1−/− (KL) mediated tumorigenesis; in contrast to intact autophagy KL lung tumors, loss of an autophagy-essential gene Atg7 dramatically impaired tumor initiation and tumor growth. This is in sharp contrast to wild-type (WT) Lkb1 (KrasG12D/+;p53−/− (KP)) tumors that are less sensitive to autophagy gene ablation. These observations indicate that LKB1 mutations predispose KRAS NSCLC to autophagy inhibition and that LKB1 mutations could be explored as a predictive biomarker for precision lung cancer therapy. To further value our discoveries in clinical translational ability, we treated mouse lung tumor-derived cell lines (TDCLs) with FDA approved autophagy inhibitor hydroxychloroquine (HCQ) and found that KL TDCLs were much sensitive to HCQ-induced cell death compared with Lkb1-WT KP TDCLs. Furthermore, a combination treatment of HCQ with mitogen-activated protein kinase kinase (MAPKK or MEK) inhibitor Trametinib showed synergistic anti-proliferative effects in KL TDCLs, but not in KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomic profiling of KL TDCLs with Trametinib, HCQ alone or in combination treatment. We found that the levels of several glycolytic and TCA cycle intermediates, as well as amino acids, were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combined treatment with HCQ and trametinib. Moreover, Trametinib treatment led to an increased level of ATP, which was decreased by the combination treatment. Similarly, we observed anti-tumor synergistic effects of the combination treatment in KL allograft tumor growth. In addition, metastases of KL TDCLs to the lung was reduced by the combination treatment compared to the single agents. We further found that in KRAS-mutant human lung cancer cell lines, LKB1-mutant cells were much sensitive to the combination treatment than LKB1-WT cells. Our observations suggest that KL TDCLs and KL human lung cancer cells resist to Trametinib treatment by upregulating autophagy to maintain energy homeostasis for survival. Therefore, combination therapy of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient NSCLC. Citation Format: Vrushank Dharmesh Bhatt, Khoosheh Khayati, Jerry Kong, Akash Raju, Zhixian Hu, Xiaoyang Su, Jessie Yanxiang Guo. Autophagy inhibition sensitizes Liver Kinase B1 (LKB1) - deficient Kras-driven lung tumors to MEK inhibitor Trametinib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5710.

Inhibition of DDR1 enhances in vivo chemosensitivity in KRAS-mutant lung adenocarcinoma.

Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive biomarkers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DDR1 is upregulated during chemotherapy both in vitro and in vivo. Moreover, analysis of a cohort of patients with LUAD suggested that high DDR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DDR1 inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD.