Non-small Cell Lung Cancer Xenograft Models Research Articles

MiR-495 and miR-5688 are down-regulated in non-small cell lung cancer under hypoxia to maintain interleukin-11 expression.

BackgroundHypoxia is a hallmark of cancer and is associated with poor prognosis. However, the molecular mechanism by which hypoxia promotes tumor progression remains unclear. MicroRNAs dysregulation has been shown to play a critical role in the tumor and tumor microenvironment. Here, we investigated the roles of miR‐495 and miR‐5688 in human non‐small cell lung cancer (NSCLC) and their underlying mechanism.MethodsThe expression levels of miR‐495 and miR‐5688 in human NSCLC tissue specimens were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR). Deferoxamine (DFO) was used to determine whether the regulation of miR‐495 and miR‐5688 under hypoxia was dependent on hypoxia‐inducible factor 1‐alpha (HIF‐1α). Furthermore, the functions of miR‐495 and miR‐5688 in tumor progression were evaluated using colony formation, 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS), wound healing, transwell assays, and xenograft model. Two algorithms, PicTAR and Targetscan, were used to predict the target gene of these two miRNAs, and dual‐luciferase reporter assay was conducted to confirm the target. The unpaired two‐tailed t test, Pearson correlation analysis, and Fisher's exact probability test were performed for statistical analyses.ResultsTwo miRNAs, miR‐495 and miR‐5688, were found to participate in NSCLC progression under hypoxia. They were down‐regulated in NSCLC tissues compared with normal tissues. We determined that hypoxia led to the down‐regulation of miR‐495 and miR‐5688 in NSCLC cells, which was independent of HIF‐1α and cellular metabolic energy. In addition, miR‐495 and miR‐5688 suppressed cell proliferation, migration, and invasion in vitro. The NSCLC xenograft model showed that miR‐495 and miR‐5688 inhibited tumor formation in vivo. Interestingly, we found that miR‐495 and miR‐5688 had the same target, interleukin‐11 (IL‐11). Recombinant human IL‐11 counteracted the effects of miR‐495 and miR‐5688 on NSCLC cells, suggesting that miR‐495 and miR‐5688 executed their tumor suppressive role by repressing IL‐11 expression.ConclusionWe found that hypoxia down‐regulated the expression levels of miR‐495 and miR‐5688 in NSCLC to enhance IL‐11 expression and tumor progression, indicating that the miR‐495/miR‐5688/IL‐11 axis may serve as a therapeutic target and potential biomarker for NSCLC.

Intrapulmonary inoculation of multicellular spheroids to construct an orthotopic lung cancer xenograft model that mimics four clinical stages of non-small cell lung cancer

IntroductionLung cancer leads in mortality among all types of cancer in US and Non-small cell lung cancer (NSCLC) is the major type of lung cancer. Mice models of lung cancer based on subcutaneous or orthotopic inoculation of cancer cell suspension do not adequately mimic the progression of lung cancer in clinic. MethodsA549-iRFP cells (human NSCLC adenocarcinoma) were cultured to form multicellular spheroids (MCS), which were then inoculated intrapulmonarily into male athymic nude mice. The xenograft cancer development was monitored by in vivo fluorescent imaging and validated by open-chest anatomy, ex vivo fluorescent imaging, and histological studies. ResultsThe newly developed orthotopic xenograft model of lung cancer simulated all four clinical stages of NSCLC progression over one month: Stage 1) localized tumor at the inoculation site, Stage 2) multiple tumor nodules or larger tumor nodule on the same side of the lung, Stage 3) cancer growth on heart surface, and Stage 4) metastatic cancer on both sides of the lung. The model yielded high rates of postoperative survival (100%) and parenchymal tumor establishment (88.9%). The roughness of the inoculated MCS associated negatively with the time needed to develop metastatic cancer (p = .0299). DiscussionThis new orthotopic xenograft model of NSCLC would facilitate the development of medications to treat lung cancer.

Downregulation of KIAA1199 by miR-486-5p suppresses tumorigenesis in lung cancer.

Lung cancer is the primary cause of death among cancer patients in China, among which nonsmall cell lung cancer (NSCLC) makes up the great majority. Hence, it is imperative to identify the biomarkers and mechanisms involved in NSCLC oncogenesis. Our present research found that KIAA1199 expression was significantly increased in NSCLC and closely related to cell proliferation, motility, and poor prognosis. We demonstrated that knockdown of KIAA1199 reduced NSCLC cell growth and motility in vitro whereas overexpression of KIAA1199 had the opposite effect. Inhibition of KIAA1199 significantly suppressed tumor growth in mouse NSCLC xenograft models. Mechanistically, as an epidermal growth factor receptor (EGFR)‐binding protein, KIAA1199 promotes EGFR signaling and regulates EGFR‐dependent Src, Erk, and Akt phosphorylation, as well as downstream kinases in the EGF‐mediated EMT pathway. We demonstrated that KIAA1199 can function as a direct binding target for miR‐486‐5p and that miR‐486‐5p overexpression can attenuate proliferation and migration of NSCLC cells via regulating the EGFR signaling pathways. To conclude, our results defined KIAA1199 as an oncogenic protein that promotes cancer cell proliferation and migration by regulating EGF‐mediated signaling pathways. This study provided new insight into NSCLC oncogenesis, which could lead to the development of innovative therapeutic plans for NSCLC.

FGFR Inhibition Enhances Sensitivity to Radiation in Non-Small Cell Lung Cancer.

FGFRs are commonly altered in non-small cell lung cancer (NSCLC). FGFRs activate multiple pathways including RAS/RAF/MAPK, PI3K/AKT, and STAT, which may play a role in the cellular response to radiation. We investigated the effects of combining the selective FGFR 1-3 tyrosine kinase inhibitor AZD4547 with radiation in cell line and xenograft models of NSCLC. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, autophagy, cell cycle, and DNA damage signaling and repair assays. In vivo xenografts and IHC were used to confirm in vitro results. NSCLC cell lines demonstrated varying degrees of FGFR protein and mRNA expression. In vitro clonogenic survival assays showed radiosensitization with AZD4547 in two NSCLC cell lines. In these two cell lines, an increase in apoptosis and autophagy was observed with combined radiation and AZD4547. The addition of AZD4547 to radiation did not significantly affect γH2AX foci formation. Enhanced xenograft tumor growth delay was observed with the combination of radiation and AZD4547 compared with radiation or drug alone. IHC results revealed inhibition of pMAPK and pS6 and demonstrated an increase in apoptosis in the radiation plus AZD4547 group. This study demonstrates that FGFR inhibition by AZD4547 enhances the response of radiation in FGFR-expressing NSCLC in vitro and in vivo model systems. These results support further investigation of combining FGFR inhibition with radiation as a clinical therapeutic strategy.

In vitro and in vivo synergistic efficacy of ceritinib combined with programmed cell death ligand-1 inhibitor in anaplastic lymphoma kinase-rearranged non-small-cell lung cancer.

Both ceritinib (CER) and programmed cell death (PD)‐1/PD ligand‐1 (PD‐L1) have brought significant breakthroughs for anaplastic lymphoma kinase (ALK)‐rearranged non‐small‐cell lung cancer (NSCLC). However, the overall clinical efficacy of either CER or PD‐1/PD‐L1 inhibitor monotherapy has been limited to a large extent. In addition, the antitumor effect of combined CER and PD‐L1 inhibitor in ALK‐rearranged NSCLC is not fully understood. In H2228 cells, we examined the tumor killing effect of CER plus PD‐L1 inhibitor in vitro by quantitative RT‐PCR, flow cytometry, ELISA, western blot analysis, PBMC coculture system, and plasmid and transfection experiments. A Ba/F3 (EML4‐ALK‐WT) xenograft mouse model was also utilized to further evaluate the synergistic anticancer effects of CER and PD‐L1 inhibitor in vivo. The coculture system of PBMCs with H2228 cells promotes the expression of PD‐L1 and phospho‐ERK, and combined treatments facilitate lymphocyte proliferation and activation, inhibit PD‐L1 expression, and enhance lymphocyte cytotoxicity and cell death. In the in vivo NSCLC xenograft model, the volumes of tumors treated with CER and PD‐L1 inhibitor in combination were significantly smaller than those treated with CER or PD‐L1 alone. The relative tumor growth inhibitions were 84.9%, 20.0%, and 91.9% for CER, PD‐L1 inhibitor, and CER plus PD‐L1 groups, respectively. Ceritinib could synergize with PD‐1/PD‐L1 blockade to yield enhanced antitumor responses along with favorable tolerability of adverse effects. Ceritinib and PD‐L1 inhibitor combined produced a synergistic antineoplastic efficacy in vitro and in vivo, which provides a key insight and proof of principle for evaluating CER plus PD‐L1 blockade as combination therapy in clinical therapeutic practice.

Aloperine in combination with therapeutic adenoviral vector synergistically suppressed the growth of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and ranked top in terms of incidence and mortality in men and women. Recently, improvements in treatment approaches for NSCLC have reported, but still, there is a need to devise innovative treatment strategies, especially to manage the advanced and metastatic stage of NSCLC. Aloperine (ALO), an herbal alkaloid, has exerted anti-cancer effects in many cancers. However, the use of any chemotherapeutic agents is dose limited due to possible adverse effects and drug-resistance issues. Therefore, a combination of chemotherapy with viral-based targeted gene therapy may provide a novel treatment strategy for NSCLC. In this study, the results of the MTT and flow cytometry-based assays showed that Aloperine-Adbic (adenoviral vector expressing p14ARF/p53) combined treatment on NSCLC cells synergistically produced anti-proliferative effects, induced apoptosis, and arrested cell cycle at the G1 phase. Furthermore, the expression analysis suggested that the p53/p21 pathway might contribute to achieving aforesaid cytotoxic effects. The ALO-Adbic combined treatment prolonged the percent survival of NSCLC xenograft models. In conclusion, ALO-Adbic combination can produce synergistic anti-cancer effects at low doses, and may offer a more effective and less toxic new treatment strategy for NSCLC.

Polyphyllin I activates AMPK to suppress the growth of non-small-cell lung cancer via induction of autophagy

Polyphyllin I (PPI), a bioactive constituent extracted from the rhizomes of Paris polyphylla, is cytotoxic to several cancer types. This study was designed to explore whether PPI prevents non-small-cell lung cancer (NSCLC) growth and to investigate the molecular mechanism. AMP-activated protein kinase (AMPK) has been implicated in the activation of autophagy in distinct tissues. In cultured human NSCLC cell lines, PPI induces autophagy by activating AMPK and then inhibiting mTOR signaling in a concentration-dependent manner. Furthermore, the activation of autophagy induced by PPI was reversed by the AMPK inhibitor compound C. Computational docking showed that PPI directly interacted with the allosteric drug and metabolite site of AMPK to stabilize its activation. Microscale thermophoresis and Drug Affinity Responsive Targeting Stability (DARTS) assay further confirmed the high affinity between PPI and AMPK. In vivo studies indicated that PPI suppressed the growth of NSCLC and increased the levels of LC3-II and phosphorylated AMPK in tumors isolated from a xenograft model of NSCLC in mice. Moreover, PPI exhibited favorable pharmacokinetics in rats. In summary, PPI conclusively acts as a direct AMPK activator to induce cell autophagy which inhibits the growth of NSCLC cells. In the future, PPI therapy should be applied to treat patients with NSCLC.

Long Non-Coding RNA HOXA11-AS Promotes Non-Small Cell Lung Cancer Tumorigenesis Through microRNA-148a-3p/DNMT1 Regulatory Axis.

ObjectiveOur present study aimed to further investigate the molecular basis of long non-coding RNA homeobox A11 antisense (HOXA11-AS) in the tumorigenesis of non-small cell lung cancer (NSCLC).MethodsHOXA11-AS, microRNA-148a-3p (miR-148a-3p), and DNA methyltransferase 1 (DNMT1) mRNA levels were measured by RT-qPCR assay. DNMT1 protein level was determined by Western blot assay. Cell proliferative capacity and apoptotic rate were determined by CCK-8 assay and flow cytometry analysis, respectively. The relationships of HOXA11-AS, miR-148a-3p, and DNMT1 were tested through bioinformatics analysis, luciferase assay, and RNA pull down assay. Mouse xenograft models of NSCLC were established to examine the biological function of HOXA11-AS in vivo.ResultsHOXA11-AS expression was notably upregulated and miR-148a-3p expression was conspicuously downregulated in NSCLC tissues and cells. HOXA11-AS knockdown curbed NSCLC cell proliferation and promoted cell apoptosis through directly increasing miR-148a-3p expression. Moreover, miR-148a-3p overexpression suppressed NSCLC cell proliferation and induced cell apoptosis. HOXA11-AS functioned as a competing endogenous RNA (ceRNA) of miR-148a-3p to increase DNMT1 expression in NSCLC cells. And, DNMT1 upregulation weakened the influence of HOXA11-AS1 loss on NSCLC cell proliferation and apoptosis. Additionally, HOXA11-AS knockdown suppressed NSCLC xenograft growth by upregulating miR-148a-3p and downregulating DNMT1 in vivo.ConclusionHOXA11-AS facilitated NSCLC tumorigenesis through miR-148a-3p/DNMT1 axis in vitro and in vivo, deepening our understanding of the molecular basis of HOXA11-AS in the development of NSCLC.

Fluzoparib increases radiation sensitivity of non-small cell lung cancer (NSCLC) cells without BRCA1/2 mutation, a novel PARP1 inhibitor undergoing clinical trials

Poly (ADP-ribose) polymerase 1 inhibitors were originally investigated as anti-cancer therapeutics with BRCA1/2 genes mutation. Here, we investigate the effectiveness of a novel PARP1 inhibitor fluzoparib, for enhancing the radiation sensitivity of NSCLC cells lacking BRCA1/2 mutation. We used MTS assays, western blotting, colony formation assays, immunofluorescence staining, and flow cytometry to evaluate the radiosensitization of NSCLC cells to fluzoparib and explore the underlying mechanisms in vitro. Through BRCA1 and RAD50 genes knockdown, we established dysfunctional homologous recombination (HR) DNA repair pathway models in NSCLC cells. We next investigated the radiosensitization effect of fluzoparib in vivo using human NSCLC xenograft models in mice. The expression of PARP1 and BRCA1 in human NSCLC tumor samples was measured by immunohistochemistry. Furthermore, we sequenced HR-related gene mutations and analyzed their frequencies in advanced NSCLC. In vitro experiments in NSCLC cell lines along with in vivo experiments using an NSCLC xenograft mouse model demonstrated the radiosensitization effect of fluzoparib. The underlying mechanisms involved increased apoptosis, cell-cycle arrest, enhanced irradiation-induced DNA damage, and delayed DNA-damage repair. Immunohistochemical staining showed no correlation between the expression of PARP1 and BRCA1. Moreover, our sequencing results revealed high mutation frequencies for the BRCA1/2, CHEK2, ATR, and RAD50 genes. The potential therapeutic value of fluzoparib for increasing the radiation sensitivity of NSCLC is well confirmed. Moreover, our findings of high mutation frequencies among HR genes suggest that PARP1 inhibition may be an effective treatment strategy for advanced non-small cell lung cancer patients.

Non-small cell lung cancer tumour antigen, MUC-1 peptide-loaded non-aggregated poly (lactide-co-glycolide) nanoparticles augmented cellular uptake in mouse professional antigen-presenting cells: optimisation and characterisation

Aim: MUC-1 lipopeptide vaccine exhibited immense potential in the treatment of non-small cell lung cancer (NSCLC) in both preclinical and clinical trials. However, it lacks triggering of mucosal immunity at the site of action. Therefore, in present investigation, MUC-1 peptide-loaded poly(lactide-co-glycolide) nanoparticles (MUC-1 peptide-PLGA-NPs) and MUC-1 peptide-loaded poly(lactide-co-glycolide) non-aggregated nanoparticles (MUC-1 peptide-PLGA-NA-NPs) using Central Composite Design (CCD) were customised.Methods and Results: The mean particle size of MUC-1 peptide PLGA-NPs was estimated to be 176.7 ± 32.7 nm, significantly (p < 0.05) higher than 100.3 ± 24.3 nm of MUC-1 peptide-PLGA-NA-NPs. Furthermore, integrity and stability of MUC-1 were maintained in MUC-1 peptide PLGA-NA-NPs. MUC-1 peptide-PLGA-NA-NPs exhibited augmented cellular uptake in mouse RAW264.7 macrophages preferably by clathrin-mediated endocytosis pathway as compared to phagocytosis followed by MUC-1-peptide PLGA-NPs owing to size ≤100 nm, and spherical shape.Conclusion: MUC-1 peptide-PLGA-NA-NPs may be a potential candidate to study antitumor potential in xenograft model of NSCLC through inhalation route of administration.

Enapotamab vedotin, an AXL-specific antibody-drug conjugate, shows preclinical antitumor activity in non-small cell lung cancer.

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

EP1.03-08 MiR-744 Facilitates Non-Small Cell Lung Cancer Progression by Transcriptional Regulation of c-FOS

Metastasis is the leading cause of lung cancer associated death. Here, we focused on the function and downstream molecular mechanism of miR-744 and its potential clinical application in non-small cell lung cancer (NSCLC) progression. The clinical cohort and data from TCGA were analyzed for the correlation of miR-744 and clinical outcomes. Multiple NSCLC cell lines and a NSCLC xenograft model were applied for the functional studies in vitro and in vivo respectively. Reporter assays were used for transcriptional regulatory mechanism study. It was confirmed that the overexpression of miR-744 was significantly correlated with lymph node metastasis and poor prognosis in NSCLC. It was an independent prognostic molecular marker for NSCLC. Both in vitro and in vivo studies revealed that miR-744 overexpression aggravated the invasion and metastasis of NSCLC cells. MiR-744 positively regulated c-FOS by directly binding to the promoter of c-FOS. We also identified -358 to -332 bp and -221 to -192 bp upstream of c-FOS gene as the direct and efficient miR-744 binding site in c-FOS promoter region. MicroRNA-744 could regulate MAPK signaling and enhanced the resistance of lung cancer cells to radiotherapy and paclitaxel. Our findings uncover the function of miR-744 in NSCLC and reveal a novel mechanism of miR-744 in mediating growth and metastasis of NSCLC cells. Our data suggests that miR-744 may serve as a possible therapeutic target for NSCLC. Support: 81572279, 2016J004, LC2016PY016, 2018CR033.

87P - Preclinical pharmacokinetic/pharmacodynamic (PK/PD) relationship of ABN401, a highly selective met inhibitor, in gastric and non-small cell lung cancer models

BackgroundABN401 is a highly selective best-in-class Met inhibitor that is targeting MET-driven cancers. Met which is also known as a hepatocyte growth factor (HGF) receptor is encoded by the MET gene. MET alterations, including amplification and mutation, and Met overexpression are well known for tumorigenic transformation, tumor growth, angiogenesis, invasion, and metastasis in several cancer types, including gastric and non-small-cell lung cancer (NSCLC). ABN401 shows significant MET signaling inhibition in in vitro and in vivo preclinical studies, especially for cell lines, xenograft, and PDx models which have high MET amplification and/or MET exon 14 deletion. MethodsIn this study, to confirm the pharmacokinetic and pharmocodynamic correlation of ABN401, gastric cancer and NSCLC xenograft models were used. SNU-5, a gastric cancer cell line and EBC-1, a NSCLC cell line both of which has high MET amplification, were used for the xenograft model and dosed at 10mg/kg and 30mg/kg of ABN401 by oral administration. Pharmacokinetic parameters were analyzed in both plasma and tumor tissue samples. Pharmacodynamic biomarkers including Met and phospho-Met (T1234/1235) and downstream signaling were analyzed by immunoblotting. In addition, an expression of Met and phospho-Met were analyzed by immunohistochemistry. ResultsThe PK results demonstrated that in both gastric and NSCLC xenograft models ABN401 drug was readily distributed to tumor tissues. According to both PD studies, ABN401 showed inhibitory effects of Met and downstream signaling in a time dependent manner for both cancer types. There was also correlated between the PK parameters in plasma and tumor samples and pharmacodynamics studies. ConclusionsThis preclinical PK/PD correlation study of ABN401 provides evidence for human dose predictions and dosing strategy for clinical studies. Legal entity responsible for the studyThe authors. FundingAbion Inc. DisclosureAll authors have declared no conflicts of interest.

Abstract 372: VGF is functionally responsible for chemotherapy resistance in non-small cell lung cancer

Abstract Despite the success of immune checkpoint therapy, most non-small cell lung cancer (NSCLC) patients still receive conventional chemotherapy. Overcoming chemotherapy resistance by identifying specific targets should improve cancer treatment and patient survival. In this study, we examined a prospective chemotherapy-resistant subpopulation in NCI-H1975 NSCLC cells and identified a novel target, neurosecretory protein VGF (VGF). Using flow cytometry, we first analyzed the expression profile of cancer stem cell markers in 100 cancer cell lines and found that NCI-H1975 cells could be divided into three subpopulations, CD44-low (CD44-L), CD44-middle (CD44-M) and CD44-high cells based on the expression pattern of CD44. CD44-M cells account for less than 5% of the NCI-H1975 cells under normal conditions, but only this subpopulation can proliferate in vitro and in vivo after treatment with cytotoxic agents, such as paclitaxel, pemetrexed and 5-fluorouracil. Affymetrix microarray analysis revealed that VGF is specifically expressed in CD44-M cells and we hypothesized that VGF would play a critical role in chemotherapy resistance. We next employed patient-derived xenograft (PDX) models of NSCLC to confirm this hypothesis with human clinical samples. NOD/SCID mice were subcutaneously transplanted with PDX and treated with either vehicle or paclitaxel (12 mg/kg, Q3Dx3, i.v.). VGF immunohistochemistry on resected PDX revealed that the number of VGF-expressing cells increased 5.6-fold in the paclitaxel-treated group compared to the vehicle control. We also performed VGF immunohistochemistry on NSCLC surgical specimens which were obtained from 127 patients, consisting of 42 neoadjuvant chemotherapy treated cases and 85 non-treated cases. The frequency of VGF-expressing specimens was higher in neoadjuvant chemotherapy treated cases (50%) than non-treated cases (18%). These results suggest that VGF-expressing tumor subpopulation is paclitaxel-resistant even in clinical settings. We also analyzed the molecular functions of VGF. Knockdown of VGF expression by siRNAs could signifıcantly suppress paclitaxel resistance in CD44-M cells. On the other hand, overexpression of VGF could significantly induce paclitaxel resistance in CD44-L cells. These results suggest that VGF is functionally responsible for chemotherapy resistance in NSCLC patients and combined administration of an anti-VGF drug and chemotherapy could be effective for preventing cancer recurrence and prolonging cancer free survival. Citation Format: Wataru Nogami, Yumi Tona, Soichi Tofukuji, Yoshino Ishioka, Mitsunobu Matsumoto, Hajime Yamada, Kenji Kuwabara, Hidekazu Tanaka, Shigeki Adachi, Yoko Yamamoto, Ryu Kanzaki, Soichiro Funaki, Yasushi Shintani, Meinoshin Okumura, Taisei Nomura. VGF is functionally responsible for chemotherapy resistance in non-small cell lung cancer [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 372.

Abstract 4415: CDK4/6 inhibition with lerociclib (G1T38) delays acquired resistance to targeted therapies in preclinical models of non-small cell lung cancer

Abstract Tyrosine kinase inhibitors (TKIs) targeting oncogenes such as EGFR, ALK, or RET have dramatically improved anti-tumor efficacy in patients with non-small cell lung cancer (NSCLC). Despite the significant clinical benefit offered by these agents, tumor responses to single-agent TKIs nevertheless remain limited in their magnitude and duration. Here we investigate combination therapy approaches with lerociclib (G1T38), a selective, oral, and potential best-in-class inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6). Consistent with previous studies reporting frequent cell-cycle gene alterations in NSCLC, we show that lerociclib synergizes with TKIs targeting EGFR, ALK, and RET in lung cancer cell lines and enhances the efficacy of osimertinib and crizotinib in PDX models harboring EGFR or ALK mutations, respectively. Moreover, in a well-characterized murine xenograft model of EGFR-mutant NSCLC, seven days of treatment with a clinically-relevant dose of osimertinib (25 mg/kg p.o. QDx7) resulted in 60% complete tumor responses, versus 100% complete tumor responses in the osimertinib plus lerociclib (25 mg/kg osi + 100 mg/kg lero, p.o. QDx7) group. Strikingly, complete tumor responses in the combination group persisted through an additional 35 days (or more) of observation, by which time all tumors in the osimertinib monotherapy group had re-emerged - suggesting a role for lerociclib in extending the duration of antitumor response. Based on this result, we assessed the ability of lerociclib to delay the emergence of resistance by chronically treating NSCLC cell lines with TKI +/- lerociclib. These experiments revealed that combination treatment with lerociclib significantly delayed acquired TKI resistance in multiple NSCLC cell lines. The same result was achieved in vivo in an EGFR-mutant NSCLC xenograft model known to develop resistance to EGFR TKI therapy, wherein combination treatment with lerociclib delayed the outgrowth of osimertinib-resistant tumors. Studies to characterize the mechanism by which lerociclib delays TKI resistance in NSCLC are ongoing. Finally, we addressed whether combination therapy with lerociclib can treat TKI resistance after it develops. In EGFR-mutant NSCLC xenografts harboring MET amplification - one of the more common mechanisms of resistance observed to EGFR TKIs in the clinic - the combination of lerociclib plus osimertinib caused significant tumor growth inhibition (90%) compared to osimertinib monotherapy (34%) after 46 days of continuous daily dosing. Collectively, our results suggest a compelling rationale for utilizing lerociclib as a backbone therapy for multiple combination regimens in NSCLC. A phase 1b/2 clinical trial evaluating lerociclib plus osimertinib in EGFR-mutant NSCLC is ongoing (NCT03455829). Citation Format: Daniel M. Freed, Claire R. Hall, Jay C. Strum, Patrick J. Roberts. CDK4/6 inhibition with lerociclib (G1T38) delays acquired resistance to targeted therapies in preclinical models of non-small cell lung cancer [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 4415.

Abstract 1236: Subclinical doses and choice of replication stress inducer in combination with CHK1 inhibitors for optimal tumor control and immune responses in vitro and in vivo

Abstract Background: Lung cancer and melanoma are responsible for almost 25% of cancer deaths in Australia. Despite recent progress with targeted and immuno-therapies, the 5 year survival rate in late stage patients is still&amp;lt;20%. CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress, primarily Gemcitiabine. Clinical trials of this combination have shown some good responses but high levels of normal tissue toxicity. Materials and methods: Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as combinations with subclinical doses of hydroxyurea (HU) in melanoma and non-small cell lung cancer (NSCLC) tumor spheres and cell lines in 2% O2 conditions and xenograft models in immune compromised mice and with humanized immune systems. Results: We report that low dose HU increased the sensitivity of &amp;gt;70% of both melanoma and NSCLC cell lines to CHK1 inhibitor (GDC-0575) triggered apoptosis, with complete loss of viability found with clinically achievable doses of this combination. Similar sensitivity was observed in xenograft models of both melanoma and NSCLC. We also demonstrate that a low dose of Gemcitabine combination with CHK1 inhibitor results in complete loss of proliferative potential in normal tissue, whereas normal tissue retaining proliferative potential after treatment with even high doses of hydroxyurea in combination with CHK1 inhibitor. In vivo, this translates to minimal effect on lymphocyte populations in the blood. The combination also triggers an inflammatory response involving the recruitment of macrophages, associated with increased HMGB1 nuclear staining. Immune responses were also assessed in humanized mouse models. Discussion: These data indicate that the combination of low dose HU and CHK1 inhibitor have strong anti-cancer activity in the setting of melanoma and NSCLC cancer, and triggers an inflammatory response, and is likely to be better tolerated than current combinations with Gemcitabine. Our data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination, and this appears to include an immune response to the tumor. Citation Format: Zay Yar Oo, Martina Proctor, Alex Stevenson, Deborah Nazareth, Jill Larsen, Nikolas Haass, Brian G. Gabrielli. Subclinical doses and choice of replication stress inducer in combination with CHK1 inhibitors for optimal tumor control and immune responses in vitro and in vivo [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 1236.

LOXL1 Is Regulated by Integrin α11 and Promotes Non-Small Cell Lung Cancer Tumorigenicity.

Integrin α11, a stromal collagen receptor, promotes tumor growth and metastasis of non-small cell lung cancer (NSCLC) and is associated with the regulation of collagen stiffness in the tumor stroma. We have previously reported that lysyl oxidase like-1 (LOXL1), a matrix cross-linking enzyme, is down-regulated in integrin α11-deficient mice. In the present study, we investigated the relationship between LOXL1 and integrin α11, and the role of LOXL1 in NSCLC tumorigenicity. Our results show that the expression of LOXL1 and integrin α11 was correlated in three lung adenocarcinoma patient datasets and that integrin α11 indeed regulated LOXL1 expression in stromal cells. Using cancer-associated fibroblast (CAF) with either a knockdown or overexpression of LOXL1, we demonstrated a role for LOXL1 in collagen matrix remodeling and collagen fiber alignment in vitro and in vivo in a NSCLC xenograft model. As a consequence of collagen reorganization in NSCLC tumor stroma, we showed that LOXL1 supported tumor growth and progression. Our findings demonstrate that stromal LOXL1, under regulation of integrin α11, is a determinant factor of NSCLC tumorigenesis and may be an interesting target in this disease.

Afatinib-loaded immunoliposomes functionalized with cetuximab: A novel strategy targeting the epidermal growth factor receptor for treatment of non-small-cell lung cancer

Afatinib, a selective and irreversible inhibitor of tyrosine kinase, was approved for the treatment of advanced non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) overexpression in 2013. Cetuximab (CTX), an anti-EGFR monoclonal antibody, is co-administered with afatinib to improve efficacy. Unfortunately, dose-related adverse reactions caused by combination therapy have affected patient compliance, and have resulted in treatment discontinuation in severe cases. In the present study, afatinib was encapsulated in “liposomes” (LPs) to achieve longer circulation in the blood and an enhanced permeability-and-retention effect in tumors. Concomitantly, CTX was designed to bind to drug-loaded LPs to form “immuno-LPs” for tumor-cell selectivity and therapeutic activity. In vitro, the cellular internalization rate of immuno-LPs was significantly higher than that of LPs (p < 0.05). In vivo, a markedly increased area under the curve and prolonged terminal half-life were detected in rats injected with the two LP formulations, indicating that LP encapsulation protected afatinib from binding to hemoglobin to control the risk of idiosyncratic drug reactions. Compared with free afatinib and LPs, immuno-LPs exhibited strongly enhanced drug delivery and antitumor efficacy in an NSCLC xenograft model, with stronger tumor selectivity and potentially fewer side-effects. Hence, EGFR-targeting immuno-LPs appear to be promising for NSCLC treatment.

KIAA1199 promotes invasion and migration in non-small-cell lung cancer (NSCLC) via PI3K-Akt mediated EMT.

KIAA1199 is often upregulated in cancer cells, including non-small-cell lung cancer (NSCLC). Although KIAA1199 is associated with aggressive tumor phenotype and poor survival in NSCLC, little is known about its functional role in NSCLC progression. Using archived clinical samples, we evaluated KIAA1199 messenger RNA (mRNA) and protein expression in NSCLC tissues and correlated with NSCLC clinicopathological characteristics as well as overall survival. Using NSCLC cell lines, KIAA1199 was either silenced using gene-specific shRNA or overexpressed to assess the impact on EMT signaling pathways. Finally, in a mouse xenograft NSCLC model, we determine the therapeutic potential of KIAA1199 repression. Our data showed that KIAA1199 was significantly upregulated in NSCLC tissues compared to adjacent normal tissues both at the mRNA (P < 0.001) and protein levels (P < 0.05). KIAA1199 overexpression is an independent prognostic marker for overall survival (HR = 1.833). In NSCLC cell lines, KIAA1199 expression directly influences the expression of EMT markers, EMT-inducing transcription factors (EMT-TFs), and EMT signaling molecules. Knocking down of KIAA1199 expression in the mouse NSCLC xenograft model significantly suppressed tumor growth and augmented the efficacy of chemotherapy (n = 5; P < 0.05). We conclude that KIAA1199 is not only a prognostic marker but a novel therapeutic target in NSCLC through regulating EMT signaling pathway. KEY MESSAGES: KIAA1199 overexpression is an independent prognostic marker in NSCLC. KIAA1199 expression directly influences the expression of EMT markers. KIAA1199 promotes invasion and migration in NSCLC via PI3K-Akt mediated EMT.

TdIF1: a putative oncogene in NSCLC tumor progression

TdT-interacting factor 1 (TdIF1) is a ubiquitously expressed DNA- and protein-binding protein that directly binds to terminal deoxynucleotidyl transferase (TdT) polymerase. Little is known about the functional role of TdIF1 in cancer cellular signaling, nor has it previously been identified as aberrant in any type of cancer. We report here for the first time that TdIF1 is abundantly expressed in clinical lung cancer patients and that high expression of TdIF1 is associated with poor patient prognosis. We further established that TdIF1 is highly expressed in human non-small cell lung cancer (NSCLC) cell lines compared to a normal lung cell line. shRNA-mediated gene silencing of TdIF1 resulted in the suppression of proliferation and anchorage-independent colony formation of the A549 adenocarcinoma cell line. Moreover, when these TdIF1-silenced cells were used to establish a mouse xenograft model of human NSCLC, tumor size was greatly reduced. These data suggest that TdIF1 is a potent regulator of lung tumor development. Several cell cycle-related and tumor growth signaling pathways, including the p53 and HDAC1/2 pathways, were identified as participating in the TdIF1 signaling network by in silico analysis. Microarray, transcriptome and protein-level analyses validated p53 and HDAC1/2 modulation upon TdIF1 downregulation in an NSCLC cellular model. Moreover, several other cell cycle regulators were affected at the transcript level by TdIF1 silencing, including an increase in CDKN1A/p21 transcripts. Taken together, these results indicate that TdIF1 is a bona fide tumor-promoting factor in NSCLC and a potential target for therapy.