Non-small Cell Lung Cancer Models Research Articles

Rev7 loss alters cisplatin response and increases drug efficacy in chemotherapy-resistant lung cancer

Cisplatin is a standard of care for lung cancer, yet platinum therapy rarely results in substantial tumor regression or a dramatic extension in patient survival. Here, we examined whether targeting Rev7 (also referred to as Mad2B, Mad2L2, and FANCV), a component of the translesion synthesis (TLS) machinery, could potentiate the action of cisplatin in non-small cell lung cancer (NSCLC) treatment. Rev7 loss led to an enhanced tumor cell sensitivity to cisplatin and dramatically improved chemotherapeutic response in a highly drug-resistant mouse model of NSCLC. While cisplatin monotherapy resulted in tumor cell apoptosis, Rev7 deletion promoted a cisplatin-induced senescence phenotype. Moreover, Rev7 deficiency promoted greater cisplatin sensitivity than that previously shown following targeting of other Pol ζ-proteins, suggesting that Pol ζ-dependent and -independent roles of Rev7 are relevant to cisplatin response. Thus, targeting Rev7 may represent a unique strategy for altering and enhancing chemotherapeutic response.

384P BLU-945, a highly potent and selective 4th generation EGFR TKI for the treatment of EGFR T790M/C797S resistant NSCLC

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR-mutant (L858R and ex19del) driven non-small-cell lung cancer (NSCLC). T790M is the most prevalent acquired EGFR resistance mutation to treatment with 1st and 2nd generation TKIs. 3rd generation TKIs, such as osimertinib, were developed to treat patients with tumors driven by this mutation. The C797S mutation is the most significant on-target resistance mechanism to osimertinib, leading to EGFR T790M/C797S double-resistant mutants. There are currently no approved targeted therapies for NSCLC patients with T790M/C797S mutations. BLU-945 is designed to target and selectively inhibit the EGFR T790M/C797S and T790M resistant mutations. BLU-945 activity on EGFR mutants and EGFR wild-type (WT) was tested in biochemical assays and cellular phosphorylation specific EGFR AlphaLisa assays. The in vivo anti-tumor activity of BLU-945 was evaluated in an NCI-H1975 cell line-derived tumor xenograft (CDX) model, as well as in osimertinib-resistant CDX- and patient-derived xenograft (PDX) models of NSCLC. BLU-945 inhibits EGFRex19del/T790M/C797S, EGFRL858R/T790M/C797S, EGFRex19del/T790M, and EGFRL858R/T790M mutants with sub-nanomolar IC50 values in an enzyme assay with a >1000-fold window over EGFRWT enzyme activity. BLU-945 achieves potent EGFR pathway inhibition in NCI-H1975 EGFRL858R/T790M, Ba/F3 EGFRL858R/T790M/C797S, and Ba/F3 EGFRex19del/T790M/C797S cell lines and a large window relative to EGFRWT inhibition. Oral administration of BLU-945 to tumor-bearing mice demonstrated potent EGFR pathway inhibition and anti-tumor activity at well-tolerated doses in the subcutaneous NCI-H1975 CDX model, and osimertinib-resistant CDX and PDX models, as well as in the intracranial luc-H1975 model of NSCLC. BLU-945 is a potent, selective, and orally available EGFR inhibitor that shows robust anti-tumor activity in osimertinib-resistant EGFR xenograft models. Because of its pharmacological activity and selectivity for mutant EGFR, BLU-945 has the potential to demonstrate activity in osimertinib-resistant EGFR-mutant NSCLC.

Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR-Dependent and EGFR-Independent Resistance Mechanisms toward Osimertinib.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non-small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. SIGNIFICANCE: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Efficient Eradication of NSCLC by Combined DNAPK Inhibition and Carbon Ion Radiotherapy via Modulation of Tumor Invasion and Microenvironment - Beyond Direct Radiosensitization

We aimed to evaluate the impact of a novel highly potent and selective serine-threonine DNA-dependent Protein Kinase inhibitor (M3814) on high-precision particle therapy of non-small cell lung cancer (NSCLC) with carbon ions (CI) and to study the effect of this combination therapy on tumor microenvironment (TME). The effectiveness of radiotherapy ± DNA-PK inhibitor (DI) was evaluated in NSCLC models in vitro and in vivo using clinical-like radiotherapy settings. Tumor growth was followed in human xenograft A549; murine syngeneic LLC and GEMM kRAS/p53 mutant s.c. NSCLC models after treatment with DI and X-rays. Additionally, irradiation with X-rays vs. CI ± DI was performed in the orthotopic LLC model and tumor response was evaluated by histopathology and transcriptome analysis. Standardized in vitro radiobiological pipeline including clonogenic survival, DNA damage response (DDR) analysis as well as functional invasion assays together with proteome profiling were performed. DDR inhibition by DI enhanced the effect of both low- and high- LET radiotherapy, i.e., X-rays and CI, respectively. Superior efficacy of CI vs. X-rays, and additional radiosensitizing effect by DI were observed in orthotopic LLC model. Histopathological analysis showed attenuation of tumor cell proliferation and epithelial–mesenchymal transition (EMT) after radiotherapy and DI, which was most apparent after CI treatment. The efficacy of combined treatment was not only reflected by impaired DDR inhibition, but also through attenuation of tumor invasion. DI affected invasion independently of DDR alteration, by decreasing AKT signaling. The relevance of DNA-PK (PRKDC) on tumor invasion was confirmed by CRISPR-Cas9 in A549 PRKDC knock out cells. Irradiation increased endostatin/COL18 production, which in turn suppressed the invasion and migration of A549 and H460 cells. While decrease of invasiveness by X-rays was dependent on the presence of endostatin, prevention of invasion by CI was less dependent on endostatin. The role of endostatin/COL18 in efficacy of radiotherapy was further confirmed in A549 COL18 knock down model. In addition to radiosensitizing and positive TME effects in the high dose region, synergistic anti-invasive activity of DI may facilitate eradication of disseminated tumor cells adjacent to gross tumor volume thereby reducing the uncertainties of underdosing in clinical target volume. Given the steep dose gradient of CI, these data provide novel rationales for widening the therapeutic window for high-precision high-LET irradiation.

Canagliflozin Mediates Effective Blockade of the mTORC1 - HIF-1a Pathway, Inhibits Growth and Enhances Non-Small Cell Lung Cancer (NSCLC) Response to Radiotherapy (RT)

Alterations in NSCLC cell metabolism contribute to rapid tumor growth, resistance to RT and poor prognosis. The mammalian Target of Rapamycin Complex 1 (mTORC1) - Hypoxia-Inducible Factor-1α (HIF-1α) pathway mediates expression of the Warburg glycolytic phenotype in NSCLC and induces RT resistance. Earlier, we showed that the new diabetes agent Canagliflozin (CANA), developed to control hyperglycemia through inhibition of the renal Na+-glucose co-transporter 2 (SGLT2), also inhibits mitochondrial complex I respiration and blocks mTORC1 signaling. In this study, we analyzed CANA’s transcriptional and post-translational effects and its regulation on RT response in preclinical models of NSCLC. Cell proliferation and clonogenic survival assays were used to assess the efficacy of CANA (0–30 μM) and RT (0–8 Gy) in adenocarcinoma (A549, H1299, H1975) and squamous cell carcinoma (SK-MES-1) NSCLC cells. Protein immunoblotting, RT-qPCR and RNAseq of A549 NSCLC cells were performed to study regulation of pathway activation and gene expression in response to CANA and RT. In-vivo effects of the two agents were examined with ectopic A549 xenografts grown in immunodeficient nude mice treated with CANA (100 mg/kg/day in chow diet) and/or RT (5 Gy). RNAseq showed that clinically achievable CANA doses (10 μM) mediate profound regulation of NSCLC cell transcriptional activity with over 100 upregulated and 80 downregulated genes. CANA significantly downregulates HIF-1α pathway-related genes that mediate HIF-1α stability and nuclear transfer, glycolysis and lipogenesis, tyrosine kinase receptor signaling and inhibition of apoptosis. CANA treatment led to activation of AMP-activated protein kinase (AMPK) and blockade of the mammalian target of rapamycin complex 1 (mTORC1) pathway. It abolished phosphorylation of the mitosis marker, histone H3. This was consistent with long-term induction of the G1/S cyclin-dependent kinase inhibitor p27kip1. Clinically relevant doses of CANA suppressed proliferation and clonogenic survival in all NSCLC cell lines but this was enhanced in the K-Ras mutant/LKB1null A549 cells. Combination index analysis indicated additivity and sensitization interaction between the CANA and RT anti-proliferative effects. In the A549 xenograft model, CANA enhanced the cytotoxic effects of RT in an additive manner. CANA mediates effective suppression of the mTORC1-HIF-1α pathway, regulates gene expression and mediates potent anti-tumor activity in NSCLC models in-vitro and in-vivo. Given that CANA is a widely-used, well-tolerated and economical agent, this work indicates strong potential for this agent to be investigated rapidly in NSCLC clinical trials in combination with RT.

SHP-2 and PDL1 Inhibition Combined with Radiotherapy Enhances Systemic Antitumor Effects in an Anti-PD1-resistant Model of Non-small-cell-lung Cancer

Immune checkpoint inhibitors such as anti-PD1/PDL1 have emerged as promising therapies for advanced non-small-cell-lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with α-PDL1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti-PD1-resistant mouse model. We treated 129Sv/Ev mice with anti-PD1-resistant 344SQ NSCLC adenocarcinoma xenografts with oral SHP099 (an SHP-2 inhibitor) combined with XRT and intraperitoneal α-PDL1. Primary tumors were treated with XRT (3 fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. In vitro experiments include: qRT-PCR, western blot, flow-cytometry, single-cell-sequence and nano-string. Results: o The Results single-cell-sequence of biopsies from advanced NSCLC patients showed that SHP-2 was significantly higher expressed in to anti-PD-1 non-responders (n = 8) compared to responders (n = 8) (P = 0.041). Then, in vitro and in vivo experiments confirmed that SHP-2 was higher expressed in PD-1 resistant NSCLC model comparing to sensitive model in qRT-PCR, western blot and flow-cytometry. o XRT in combination with SHP099 and α-PDL1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. o Expression analysis of 770 immune-related genes indicated that triple therapy significantly increased macrophage function (P = 0.008 vs XRT alone, P = 0.028 vs XRT+α-PDL1), T-cell function (P = 0.003 vs control, P = 0.009 vs XRT alone), and decreased cancer progression (P = 0.048 vs control) in abscopal tumor sites. o Flow-cytometry immune profiling indicated that addition of anti-SHP-2 was associated with higher M1/M2 ratio, greater numbers of CD8+ T cells, and fewer regulatory T cells, compared to iRT group (XRT+anti-PD-L1). o The depletion experiment showed that the anti-tumor effects of triple therapy took place mainly through M1 TAMs and CD8+ T cells. This novel triple-combination therapy had strong antitumor effects in this mouse model of anti-PD1-resistant NSCLC, and may be a novel therapeutic approach for anti-PD1- resistant NSCLC in patients.

Targeted Radionuclide Therapy in Patient-Derived Xenografts Using 177Lu-EB-RGD.

Currently, most patients with non-small cell lung cancer (NSCLC) are diagnosed in advanced stages with a poor five-year survival rate. Therefore, intensive research aimed at finding novel therapeutic strategies has been ongoing; experimental models that reliably emulate NSCLC disease are greatly needed to predict responses to novel therapeutics. Therefore, we developed patient-derived xenograft (PDX) models of NSCLC, which we then used to evaluate the therapeutic efficacy of 177Lu-EB-RGD, a peptide-based radiopharmaceutical with improved pharmacokinetics that targets integrin αvβ3 In this study, three different groups of NSCLC-PDXs were successfully established, all of which maintained the same IHC and genetic characteristics of the human primary tumor. The two NSCLC-PDX groups with intense and low expression of integrin αvβ3 (denoted as PDXαvβ3+ and PDXαvβ3-) were chosen as the experimental models to evaluate the in vivo biological behavior of 177Lu-EB-RGD. In SPECT imaging and biodistribution studies, 177Lu-EB-RGD showed significantly higher accumulation in PDXαvβ3+ and PDXαvβ3- models than its corresponding monomer 177Lu-RGD. A single dose of 18.5 MBq 177Lu-EB-RGD was enough to completely eradicate the tumors in PDXαvβ3+, with no sign of tumor recurrence during the observation period. Such treatment was also efficacious in PDXαvβ3-: a single dose of 29.6 MBq 177Lu-EB-RGD led to a significant delay in tumor growth as compared with that in the control or 177Lu-RGD group. The preclinical data from the use of this model suggest that 177Lu-EB-RGD may be an effective treatment option for NSCLC and should be further evaluated in human trials.

Serum microRNA-365 suppresses non-small-cell lung cancer metastasis and invasion in patients with bone metastasis of lung cancer.

ObjectiveIn the present investigation, we evaluated the effects of microRNA-365 (miR-365) on non-small-cell lung cancer (NSCLC) cell metastasis and invasion in patients with bone metastasis of lung cancer.MethodsBlood samples from patients with NSCLC and healthy controls and the A549 adenocarcinoma cell line were included in this study. Quantitative real-time PCR and microarray were performed on blood samples. The MTT assay, luciferase reporter assay, Transwell assay, ELISA, and western blot were performed to evaluate expression of associated factors.ResultsExpression of miR-365 was reduced in patients with bone metastasis of NSCLC. Downregulation of miR-365 promoted cell growth, metastasis, and invasion of NSCLC. Upregulation of miR-365 reduced cell growth, metastasis, and invasion of NSCLC. Downregulation of miR-365 induced expression of NKX homeobox-1 (NKX2-1), epidermal growth factor receptor (EGFR), phosphoinositide-3-kinase (PI3K), and p-Akt proteins in an in vitro model of NSCLC. Inhibition of NKX2-1 reduced the effects of miR-365 on cell growth, metastasis, and invasion of NSCLC. Activation of EGFR reduced the effects of miR-365 on cell growth, metastasis, and invasion of NSCLC.ConclusionsThe study established that the serum miR-365 suppresses NSCLC cell metastasis and invasion in patients with bone metastasis of lung cancer via EGFR/PI3K through NKX2-1.

A new role for circulating T follicular helper cells in humoral response to anti-PD-1 therapy

BackgroundLung cancer is one of the most frequent malignancies in humans and is a major cause of death. A number of therapies aimed at reinforcing antitumor immune response, including antiprogrammed...

Abstract 4120: ORIC-101 comprehensively inhibits glucocorticoid pathways to overcome therapeutic resistance in pan-cancer models

Abstract Preclinical studies have established a role for the Glucocorticoid Receptor (GR) in mediating resistance to both anti-hormonal therapies and conventional chemotherapies in epithelial cancers. Studies in breast cancer models have established that the chemoprotective effect afforded by GR activation is facilitated, at least partially, through upregulation of genes involved in anti-apoptosis, epithelial-to-mesenchymal transition (EMT), and metastasis - thus suggesting that GR inhibition may enhance therapeutic responses. A novel GR antagonist, ORIC-101, is in early clinical development in combination with nab-paclitaxel in advanced solid tumors (NCT03928314). We set out to understand the transcriptional consequences of activating GR in a panel of 9 triple negative breast cancer (TNBC), 12 non-small cell lung cancer (NSCLC) and 12 pancreatic ductal adenocarcinoma (PDAC) cell lines, covering three indications likely to be encountered in clinical trials. Cell lines were selected to capture the transcriptional variability across subtypes of each indication, and transcriptome profiling using RNA-sequencing was performed on these cell lines after treatment with the synthetic glucocorticoid dexamethasone (Dex) in the presence or absence of ORIC-101. We present a comprehensive evaluation of GR-mediated signaling across 3 key indications and 33 cancer cell lines. Overall, Dex resulted in transcriptional regulation of hundreds of genes. While there was a high degree of variation in the specific genes regulated by GR across TNBC, NSCLC, and PDAC models, a set of 11 genes emerged as consistently upregulated by GR, termed the “GR activation signature”. The addition of ORIC-101 completely reverted the expression of these 11 signature genes back to vehicle condition levels. We further validated a subset of signature genes as ORIC-101 pharmacodynamic biomarkers that may be used to inform target engagement status within a patient population in clinical trials. Despite the high degree of variability in the GR-driven transcriptional programs at the gene level, pathway enrichment analysis revealed biological convergence towards a common set of GR regulated pathways including EMT, apoptosis, stemness, hypoxia, inflammation/immune regulation, and extracellular matrix. These molecular processes associated with GR were observed across TNBC, NSCLC, and PDAC cell lines, suggesting a common drug resistance mechanism of GR across the cancer types. Importantly, ORIC-101 completely reversed these Dex-induced changes at both the gene and the pathway level. Our results not only elucidate the direct mechanism of GR-mediated chemotherapy resistance but also showcase the ability of ORIC-101 to revert all transcriptional changes caused by GR activation. Citation Format: Aleksandr Pankov, Haiying Zhou, Shravani Barkund, Ganapati Hegde, Padmini Narayanan, Omar Kabbarah, Lori S. Friedman, Anneleen Daemen. ORIC-101 comprehensively inhibits glucocorticoid pathways to overcome therapeutic resistance in pan-cancer models [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 4120.

Abstract 5220: Development of first-in-class small molecule inhibitors of FLIP which activate caspase-8, the nodal regulator of apoptosis, necroptosis and pyroptosis

Abstract Caspase-8 is the initiator caspase for the extrinsic apoptotic pathway and now recognized as the molecular “switch” that controls 3 major forms of programmed cell death: apoptosis, necroptosis and pyroptosis. As such, methods to selectively activate caspase-8 in appropriate disease contexts represent an exciting new therapeutic paradigm. In cancer, evasion of cell death is a fundamental cause of resistance to therapy, prompting the development of therapeutics that reactivate cell death pathways, with the first of these (targeting the intrinsic apoptotic pathway) recently clinically approved. The apoptosis modulator FLIP is a non-redundant inhibitor of caspase-8 activation in the extrinsic apoptotic pathway and is the only human pseudo-caspase. FLIP is frequently overexpressed in a number of cancers and leukemias and, through its modulation of caspase-8, has been shown to be a major mediator of resistance to standard-of-care chemotherapies, targeted therapies and radiotherapy. Moreover, by regulating caspase-8 activity, FLIP is a key determinant of cell death induced by immune effector cells. Thus, targeting FLIP's interaction with caspase-8 represents a unique therapeutic approach for enhancing standard-of-care anti-cancer therapies and promoting anti-tumor immunity. We have identified a series of potent (nM), selective small molecule inhibitors which, by disrupting the binding of FLIP to effector proteins, are able to activate caspase-8-dependent apoptosis in multiple cancer cell lines. These protein-protein interaction inhibitors, which are relatively low MW and have favorable drug-like properties, have potent in vitro activity as single agents in several clinically-relevant settings, including KRAS mutant non-small cell lung cancer (NSCLC). Moreover, these FLIP inhibitors have been shown to potentiate the effects of standard-of-care chemotherapeutics and targeted anti-cancer agents in pre-clinical models. Recently, FLIP has emerged as a key mediator of resistance to the 3rd generation EGFR inhibitor Osimertinib in EGFR mutant NSCLC. We found that Osimertinib-resistant EGFR mutant NSCLC cells express elevated levels of FLIP and are more sensitive to FLIP inhibitors. Most importantly, compared to treatment with Osimertinib alone, co-treatment with Osimertinib and FLIP inhibitors prevented tumor regrowth in vivo after treatment cessation. Similar effects have been observed in vitro with the MEK inhibitor Selumetinib in KRAS mutant NSCLC models. In summary, we have identified novel, drug-like activators of caspase-8 with a unique mechanism-of-action and successfully completed proof-of-concept in vivo efficacy studies which have strong translational relevance to the clinical situation in EGFR mutant NSCLC, where despite excellent response rates to Osimertinib, patient relapse is inevitable. Moreover, these compounds have potential for broad application in treatment of several cancers both as single agents and in combination with standard-of-care therapy and other clinically-relevant targeted agents. Citation Format: Daniel B. Longley, Catherine Higgins, Jennifer Fox, Jamie Z. Roberts, Ray Boffey, Sophie Williams, Trevor Perrior, Martin J. Page, Tim Harrison. Development of first-in-class small molecule inhibitors of FLIP which activate caspase-8, the nodal regulator of apoptosis, necroptosis and pyroptosis [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 5220.

Abstract 4455: Combination of bempegaldesleukin and anti-CTLA-4 prevents metastatic dissemination after primary surgery or radiation therapy in a preclinical model of non-small cell lung cancer

Abstract Purpose: Surgical resection or stereotactic ablative radiation therapy (SBRT) in early stage non-small cell lung cancer (NSCLC) have local control rates of 90% or higher. Current treatment paradigms effectively control primary tumor sites, but distant relapse remains a challenge with metastatic failure in more than 30% of patients with primary tumors larger than 5 cm. We hypothesized that adjuvant immune checkpoint blockade with anti-CTLA4 (C4) and /or bempegaldesleukin (NKTR-214), a CD122-preferential pegylated-interleukin-2 (IL2) pathway agonist,in combination with primary tumor ablative radiation or surgical resection would reduce metastases in a spontaneously metastatic syngeneic Lewis Lung Carcinoma (LLC) model. Methods: LLC flank tumors were established in C57BL/6 mice (n=6 per replicate, 12 total). When tumors were ~100 mm3 they were randomized to receive: vehicle only (VO); NKTR-214 (16 μg on Days 6, 15, 24) + C4 (200 μg IP on Days 4, 7, 10); RT (8Gy x 3 on Days 1, 2, 3); RT + C4, RT + NKTR-214; RT + C4 + NKTR-214; Primary surgery (Day 16), or Surgery + NKTR-214 + C4. Primary tumor size, survival, and a binary assessment of metastatic disease was made for each mouse. Flow cytometry studies examined tumor immune cell infiltrates on Day 21 in mice treated with VO, RT, RT + C4, C4 + NKTR-214, or RT + C4 + NKTR-214. Results: NKTR + C4 prevented development of spontaneous metastasis (p < 0.01) but did not control primary disease compared to VO treatment. RT alone significantly reduced primary tumor size compared to VO control (p < 0.01), but these mice all developed metastases. Adjuvant NKTR-214 + C4 after RT reduced development of spontaneous metastasis in mice treated by this regimen by 91% and 73% compared to RT alone and RT + C4 respectively. RT + C4 + NKTR-214 also significantly improved survival compared to both RT + C4 and RT + NKTR-214 (p = 0.02, p = 0.003). 58% of mice treated with RT + C4 + NKTR-214 cleared their primary tumors compared to 16% with RT + C4 and 0% in RT + NKTR-214 at day 30 (p = 0.03, p < 0.01). All mice clearing primary tumors rejected LCC rechallenge. Flow cytometric immuneprofiling of the primary tumors demonstrated increased CD8+T cells, NK cells (NK1.1+CD3−), and NKT cells (NK1.1+CD3+) with NKTR-214 + C4 w/wo RT treatment compared to VO controls. NKTR-214 + C4 prior to surgical resection also reduced the development of spontaneous lung metastases compared to surgery alone (p < 0.01). Conclusions: Addition of systemic NKTR-214 + C4 treatment to RT or primary surgical resection reduces development of distant metastatic disease in a preclinical NSCLC model. Moreover, treatment with NKTR-214 + C4 increases infiltration of innate and adaptive immune effector cells in the primary tumor. This treatment strategy has important translational potential to prevent distant relapse in patients undergoing definitive local therapies for early stage NSCLC. Citation Format: Ryan J. Brown, Luke Zangl, Ian Arthur, Alex Pieper, Peter M. Carlson, Juliana Castillo, Paul M. Sondel, Alexander Rakhmilevich, Zach S. Morris, Ravi B. Patel. Combination of bempegaldesleukin and anti-CTLA-4 prevents metastatic dissemination after primary surgery or radiation therapy in a preclinical model of non-small cell lung cancer [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 4455.

Abstract 6648: Racotumomab and PD-1 blockade combination exhibits an additive antitumor effect in a non-small cell lung cancer model

Abstract N-glycolylneuraminic acid (NeuGc) is a sialic acid molecule present in mammalian cells as terminal constituents of membrane glycoconjugates such as gangliosides and glycoproteins. Although the role of NeuGc-containing glycoconjugates in human cancer is still under investigation, gangliosides such as NeuGcGM3 has been consistently reported as a tumor antigen in various epithelial and neuroectodermal malignancies, including non-small cell lung cancer (NSCLC). Interestingly, NeuGc-glycoconjugates are abundant in aggressive neoplasms, but they are usually absent in normal human tissues. In contrasts to most mammals, human beings lack the key enzyme that catalyzes N-glycolylation of sialic acid. Cancer cells can favor NeuGc intake from diet sources, thus allowing the expression of NeuGc-containing glycoconjugates. Racotumomab is an anti-NeuGc anti-idiotype monoclonal antibody that has been approved in Latin American countries as maintenance immunotherapy for advanced NSCLC. Combinatorial approaches involving long-term immunization against tumor-specific antigens together with the anti-PD1 immune-checkpoint inhibitor pembrolizumab is an attractive strategy for immunotherapy. In this work, we have examined the antitumor activity of racotumomab in combination with anti-PD1 therapy, using the 3LL Lewis lung carcinoma as a preclinical model of NSCLC in C57BL/6 mice. Immunization with either weekly or biweekly s.c. doses of racotumomab at 50-200 μg/dose formulated in aluminum hydroxide (racotumomab-alum) demonstrated a significant antitumor effect against the progression of lung tumor nodules (p<0.05, ANOVA followed by Tukey). Similarly, checkpoint blockade with an anti-mouse PD1 monoclonal antibody injected i.p. at 200 μg/dose exerted a comparable antitumor effect in this 3LL lung model (p<0.01, unpaired t-test). Interestingly, sequential administration of anti-PD1 therapy followed by repeated immunizations with racotumomab-alum was highly effective against lung nodules and well tolerated, showing a reduction in nodules formation of 62 and 45% compared to anti-PD1 or racotumomab-vaccinated groups, respectively (p<0.01, ANOVA followed by Tukey). Our preclinical data provide support for the combination of anti-PD1 checkpoint blockade with the anti-idiotype monoclonal antibody racotumomab in advanced NSCLC, since combination treatment has a significant additive antitumor effect compared to each individual treatment. Citation Format: Valeria I. Segatori, Cynthia A. Gulino, Carla S. Capobianco, Selene Rojo, Gretel M. Ferreira, Héctor A. Cuello, Mariano R. Gabri, Daniel A. Alonso. Racotumomab and PD-1 blockade combination exhibits an additive antitumor effect in a non-small cell lung cancer model [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 6648.

Abstract 1864: Generation of a triple-mutation (T790M/C797S/L858R) NSCLC cell line for relevant drug discovery and development

Abstract Epidermal Growth Factor Receptor (EGFR) overexpression is observed in a large subset of patients with non-small-cell lung cancer (NSCLC) and correlated with poor prognosis. Current approved treatment for EGFR mutation-positive NSCLC has been revolutionized with three generations of EGFR tyrosine kinase inhibitors (TKIs): the first- and second-generation TKIs, such as gefitinib, erlotinib and afatinib, selectively targeting EGFR activating mutations existing in exon 19 and 21; and the third-generation EGFR-wild-type sparing, irreversible activating mutations/T790M inhibitor, osimertinib. Patients with somatic activating mutations in the EGFR gene have dramatic response initially, but would eventually develop resistance to these TKIs. One leading evidence of osimertinib resistance is mediated by occurrence of the point mutation C797S. There are no effective therapeutic strategies to overcome this triple-mutation (activating mutations/T790M/C797S) mediated resistance. Therefore, it is urged to develop new drug to target this particular mutant. There is still lack of triple-mutation in vitro model for the new generation drug discovery. In this study, a triple-mutation NSCLC model was generated in the NCI-H1975 lung cancer cell line, which harbors other naturally occurring EGFR genomic aberrations - double-mutation (T790M/L858R) - inherent in NSCLC. We utilized the CRISPR/Cas9 genome editing tool to target endogenous loci in human NSCLC cell line NCI-H1975 and created the intend point mutation, resulting in the triple mutation cell line (T790M/C797S/L858R). Further phenotypic analysis demonstrated that the triple-mutation cell line was resistant to the third generation inhibitors in contrast to the parental double-mutation cell line. This newly developed triple-mutation lung cancer cell line provided a very useful tool for oncology drug discovery for NSCLC. Citation Format: Huiying Ma, Peixue Li, Peiran Zhang, Fuyun Sun, Qing Lin. Generation of a triple-mutation (T790M/C797S/L858R) NSCLC cell line for relevant drug discovery and development [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 1864.

Abstract CT127: A phase I study of cMET inhibitor bozitinib in patients with advanced NSCLC harboring cMET alterations

Abstract Introduction: Cellular mesenchymal-epithelial transition (cMET) dysregulation has been described in non-small-cell lung cancer (NSCLC) and implicated as a negative prognostic factor. It can occur as amplification, overexpression or mutations leading to exon 14 skipping. Bozitinib (PLB-1001, CBT-101) is a potent highly selective cMET inhibitor, which has demonstrated superior activity in both in vitro and in vivo NSCLC models. Methods: This was a phase I, open-label multicenter study conducted in locally advanced or metastatic NSCLC patients that included a dose-escalation (19 patients) and a dose-expansion phase (18 patients) (NCT02896231). A traditional 3+3 design was adopted for dose escalation. The primary objective was to characterize the safety and tolerability of single agent Bozitinib. The secondary objectives included pharmacokinetics (PK) analysis and antitumor activity assessed by RECIST v1.1. cMET dysregulation was defined by: IHC 3+ expression in >50% of tumor cells or gain of copy numbers (GCN) ≥5 or cMET/centromere ≥2.2 by FISH or MET copy number ≥2.25 or with exon 14 skipping by next-generation sequencing (NGS). Only central laboratory testing was acceptable. Patients≥18 years with locally advanced or metastatic NSCLC with an ECOG performance status ≤2 were enrolled. Those previously treated with cMET inhibitor or HGF targeting therapy were excluded. Patients were orally administrated with Bozitinib capsules in a total of 6 dose cohorts: 5 BID dose cohorts (50 mg BID, 100 mg BID, 150 mg BID, 200 mg BID and 275 mg BID), and 1 QD dose cohort (300 mg QD). The enrollment had been completed by October 31 2019 with 37 enrolled patients. Results: Of the 37 enrolled patients, with a median age of 61: 8 had overexpression; 11 had exon 14 skipping; 8 had amplification and 10 patients had more than 1 type of MET alterations. Bozitinib was generally well-tolerated in all cohorts. Treatment-related AEs of any grade were observed in 35 patients and those of ≥ grade 3 were observed in 10 patients (in 200mg BID, 275mg BID and 300mg QD groups), including ALT increase, AST increase, bilirubin increase and peripheral edema. Common AEs occurring in more than 20% of patients included: ALT increase (40.5%), AST increase (40.5%), bilirubin increase (40.5%), peripheral edema (32.4%) and QTc interval prolongation (18.9%). Bozitinib showed good linear PK characteristics, with dose-dependent increases in exposure and a half-life around 13.8-44.6h. 36 were included in the BOR analysis. One patient was excluded due to the development of brain metastases after first dose. This cohort presented an ORR of 30.6% (11/36) and a DCR of 94.4% (34/36). Sub-analysis of ORR for patients with overexpression, amplification and exon 14 skipping were 35.7%, 41.2% and 66.7%, respectively. ORR for patients with overexpression and amplification (N=6) was 50%. ORR for patients harboring both exon 14 skipping and amplification (N=4) was 100%. Conclusions: Based on safety profiles, PK and efficacy analyses, 200mg BID was determined to be RP2D. Bozitinib was well-tolerated with manageable safety profiles when administered at RP2D. Preliminary antitumor activity was observed in patients with exon 14 skipping and/or amplification, particularly in those with NGS-identified exon 14 skipping. Citation Format: Jinji Yang, Qing Zhou, Huajun Chen, Lin Wu, Jun Zhao, Renhua Guo, Yun Fan, Hepeng Shi, Weizhe Xue, Peilong Zhang, Han-Zhang Han, Xuan Lin, Shuyin Chen, Lu Zhang, Hao Liu, Xinru Mao, Bin Gan, Yilong Wu. A phase I study of cMET inhibitor bozitinib in patients with advanced NSCLC harboring cMET alterations [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 CT127.

CircRNA_102179 promotes the proliferation, migration and invasion in non-small cell lung cancer cells by regulating miR-330-5p/HMGB3 axis

Non-small cell lung cancer (NSCLC) accounting for 85 % of all lung cancer was one of the main causes of death worldwide. In this study, we investigated the role of circRNA_102179 in NSCLC development. The levels of circRNA_102179 in NSCLC tissues and cell lines were determined by quantitative real-time PCR assay (qRT-PCR). CCK8 and colony formation assays were applied to explore the effect of circRNA_102179 on the growth of NSCLC cells in vitro. Transwell assay was utilized to analyze the impact of circRNA_102179 on the migration and invasion of NSCLC cells. Target prediction and luciferase reporter assay were used to identify the interacting miRNA of circRNA_102179. The interaction among circRNA_102179/ miR-330−5p/HMGB3 was further validated by colony formation and Transwell invasion assays. Finally, the mouse xenograft NSCLC model was used to explore the role of circRNA_102179 in the tumor growth of NSCLC cells in vivo. CircRNA_102179 was overexpressed in NSCLC tissues and cells compared with normal lung tissues and human bronchial epithelial cells (HBEs). The down-regulation of circRNA_102179 markedly reduced the proliferation, migration, and invasion of NSCLC cells. Moreover, down-expression of circRNA_102179 significantly increased the level of miR-330−5p/HMGB3 in NSCLC cells. Further functional experiments indicated that over-expression of miR-330−5p reversed the inhibitory effect of circRNA_102179 on NSCLC cells growth, migration, and invasion. Our results reveal that circRNA_102179 facilitates the proliferation, migration, and invasion of NSCLC cell via modulating miR-330−5p/ HMGB3 axis in NSCLC cells.

Palbociclib treatment alters nucleotide biosynthesis and glutamine dependency in A549 cells

BackgroundAberrant activity of cell cycle proteins is one of the key somatic events in non-small cell lung cancer (NSCLC) pathogenesis. In most NSCLC cases, the retinoblastoma protein tumor suppressor (RB) becomes inactivated via constitutive phosphorylation by cyclin dependent kinase (CDK) 4/6, leading to uncontrolled cell proliferation. Palbociclib, a small molecule inhibitor of CDK4/6, has shown anti-tumor activity in vitro and in vivo, with recent studies demonstrating a functional role for palbociclib in reprogramming cellular metabolism. While palbociclib has shown efficacy in preclinical models of NSCLC, the metabolic consequences of CDK4/6 inhibition in this context are largely unknown.MethodsIn our study, we used a combination of stable isotope resolved metabolomics using [U-13C]-glucose and multiple in vitro metabolic assays, to interrogate the metabolic perturbations induced by palbociclib in A549 lung adenocarcinoma cells. Specifically, we assessed changes in glycolytic activity, the pentose phosphate pathway (PPP), and glutamine utilization. We performed these studies following palbociclib treatment with simultaneous silencing of RB1 to define the pRB-dependent changes in metabolism.ResultsOur studies revealed palbociclib does not affect glycolytic activity in A549 cells but decreases glucose metabolism through the PPP. This is in part via reducing activity of glucose 6-phosphate dehydrogenase, the rate limiting enzyme in the PPP. Additionally, palbociclib enhances glutaminolysis to maintain mitochondrial respiration and sensitizes A549 cells to the glutaminase inhibitor, CB-839. Notably, the effects of palbociclib on both the PPP and glutamine utilization occur in an RB-dependent manner.ConclusionsTogether, our data define the metabolic impact of palbociclib treatment in A549 cells and may support the targeting CDK4/6 inhibition in combination with glutaminase inhibitors in NSCLC patients with RB-proficient tumors.

Resveratrol Suppresses Tumor Progression via Inhibiting STAT3/HIF-1α/VEGF Pathway in an Orthotopic Rat Model of Non-Small-Cell Lung Cancer (NSCLC).

BackgroundThe STAT3/HIF-1α/VEGF pathway is associated with the development and progress of various tumors including NSCLC. The aim of the present study was to investigate whether resveratrol (RES) could suppress NSCLC progression via inhibiting the expressions of STAT3, HIF-1α, and VEGF in a nude rat model.MethodsTwenty-four nude rats were randomly divided into control, NSCLC, and NSCLC+RES groups. An orthotopic rat model of NSCLC was established. The animals in the NSCLC+RES group received the same operation as the NSCLC group and were intragastrically administered RES at 250 mg/kg/day for 12 weeks. Lung tissue samples were harvested for gross tumor burden measurement, histological examinations, RT-PCR, and Western blot assays.ResultsIn the NSCLC+RES group, significant decreases in lung weight index, lung tumor burden, STAT3/HIF-1α/VEGF mRNA, and protein levels were observed when compared with the NSCLC group (all P<0.05). The structural integrity of the lung was less affected and the apoptotic index was significantly higher in the NSCLC+RES group, when compared to the NSCLC group (P<0.05).ConclusionRES suppresses NSCLC partly through inhibiting the expressions of STAT3, HIF-1α, and VEGF. The STAT3/HIF-1α/VEGF pathway might be a candidate drug target for developing new chemotherapy agents derived from RES for the treatment of NSCLC.

A synthetic coumarin derivative (4-flourophenylacetamide-acetyl coumarin) impedes cell cycle at G0/G1 stage, induces apoptosis, and inhibits metastasis via ROS-mediated p53 and AKT signaling pathways in A549 cells.

New chemotherapeutic agents with minimum side effects are indispensable to treat non-small-cell lung cancer (NSCLC) since the mortality rate of patients suffering from NSCLC remains high despite receiving conventional medication. In our previous study, many coumarin derivatives were screened for their anticancer properties in A549, an in vitro NSCLC model. One of these, 4-flourophenylacetamide-acetyl coumarin (4-FPAC), induced cytotoxicity at a concentration as low as 0.16 nM. Herein, initially, the cytotoxic potential of 4-FPAC was tested on a noncancerous cell line NIH3T3 and was found safe at the selected dose of 0.16 nM. Further, we investigated the mechanism by which 4-FPAC induced cytotoxicity and arrested the progression of cell cycle as well as metastasis in A549. Results of ethidium bromide/acridine orange (EtBr/AO), 4,6-diamidino-2-phenylindole, comet, and lactate dehydrogenase assays revealed that 4-FPAC caused cytotoxicity via reactive oxygen species-induced p53-mediated mechanism, which involves both extrinsic and intrinsic pathways of apoptosis. Dichlorodihydrofluorescein diacetate, rhodamine 123, and AO staining confirmed the involvement of both mitochondria and lysosome in inducing apoptosis. However, flow cytometric analysis revealed that it causes cell cycle arrest at the G0/G1 phase by modulating p21, CDK2, and CDK4 expression. Aggregation, soft-agar, clonogenic, and scratch assays as well as gene expression analysis collectively confirmed that 4-FPAC minimizes the metastatic property of A549 by downregulating Snail, matrix metalloproteinase 9, and interleukin-8. Additional studies reaffirmed the above findings and substantiated the role of PI3K/AKT in achieving them. The cell-type-specific selective cytostatic and antimetastatic properties shown by 4-FPAC indicate its potential to emerge as a drug of choice against NSCLC in the future.

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.