Human Non-small Cell Lung Cancer Xenograft Model Research Articles

Zongertinib (BI 1810631), an irreversible HER2 TKI, spares EGFR signaling and improves therapeutic response in preclinical models and patients with HER2-driven cancers.

Mutations in HER2 occur in 2-4% of non-small cell lung cancer (NSCLC) and confer poor prognosis. ERBB-targeting tyrosine kinase inhibitors, approved for treating other HER2-dependent cancers, are ineffective in HER2 mutant NSCLC due to dose-limiting toxicities or suboptimal potency. We report the discovery of zongertinib (BI 1810631), a covalent HER2 inhibitor. Zongertinib potently and selectively blocks HER2, while sparing EGFR, and inhibits the growth of cells dependent on HER2 oncogenic driver events, including HER2-dependent human cancer cells resistant to trastuzumab deruxtecan. Zongertinib displays potent anti-tumor activity in HER2-dependent human NSCLC xenograft models and enhances the activities of antibody-drug conjugates and KRASG12C inhibitors, without causing obvious toxicities. The preclinical efficacy of zongertinib translates in objective responses in patients with HER2-dependent tumors, including cholangiocarcinoma (SDC4-NRG1 fusion) and breast cancer (V777L HER2 mutation) thus supporting the ongoing clinical development of zongertinib.

Combined inhibition of MET and VEGF enhances therapeutic efficacy of EGFR TKIs in EGFR-mutant non-small cell lung cancer with concomitant aberrant MET activation

Abstract Background Aberrant activation of mesenchymal epithelial transition (MET) has been considered to mediate primary and acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, mechanisms underlying this process are not wholly clear and the effective therapeutic strategy remains to be determined. Methods The gefitinib-resistant NSCLC cell lines were induced by concentration increase method in vitro. Western blot and qPCR were used to investigate the relationship between MET and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway. Double luciferase reporter gene and co-immunoprecipitation were used to further reveal the regulation mechanism between MET and VEGF/VEGFR2. The effect of combined inhibition of MET and VEGF/VEGFR2 signaling pathway on the therapeutic sensitivity of EGFR-TKI in gefitinib resistant cell lines with MET aberration was verified ex vivo and in vivo. Results We successfully obtained two gefitinib-resistant NSCLC cell lines with EGFR mutation and abnormal activation of MET. We observed that MET formed a positive feedback loop with the VEGF/VEGFR2 signaling, leading to persistent downstream signaling activation. Specifically, MET up-regulated VEGFR2 expression in a MAPK/ERK/ETS1-dependent manner, while VEGF promoted physical interaction between VEGFR2 and MET, thereby facilitating MET phosphorylation. A MET inhibitor, crizotinib, combined with an anti-VEGF antibody, bevacizumab, enhanced the sensitivity of NSCLC cells to gefitinib and synergistically inhibited the activation of downstream signaling in vitro. Dual inhibition of MET and VEGF combined with EGFR TKIs markedly restrained tumor growth in both human NSCLC xenograft models and in an EGFR/MET co-altered case. Conclusions Our work reveals a positive feedback loop between MET and VEGF/VEGFR2, resulting in continuous downstream signal activation. Combined inhibition of MET and VEGF/VEGFR2 signaling pathway may be beneficial for reversing EGFR TKIs resistance.

Abstract 3259: TAS3351 is a 4th-generation EGFR-TKI overcoming T790M and C797S-mediated resistance in NSCLC with EGFR common mutations

Abstract Background: Common mutations (exon 19 deletion; ex19del or L858R point mutation; L858R) of the epidermal growth factor receptor (EGFR) are found in patients with non-small cell lung cancer (NSCLC) and are sensitive to EGFR-tyrosine kinase inhibitors (TKIs), including osimertinib, a third-generation, irreversible EGFR-TKI. Although osimertinib is widely utilized as the standard of care in first-line therapy of NSCLC, resistance inevitably occurs during the treatment. Among EGFR-dependent/on-target resistant mechanisms of osimertinib, the C797S mutation is reported to emerge as the most frequent alteration. The cysteine residue at position 797 of EGFR is covalently bound with osimertinib, and its alteration to serine causes an inability to form a covalent bond, making osimertinib resistant to NSCLC with a C797S mutation. While first-generation, reversible EGFR-TKIs (gefitinib and erlotinib) might be effective for patients with C797S mutation, the T790M mutation is reported to emerge in almost half of patients with EGFR common mutations during treatment with first-generation EGFR-TKIs. Here, we report TAS3351 as a novel, next-generation EGFR-TKI designed to overcome C797S and T790M-mediated resistance in NSCLC patients. Methods: The inhibitory potency of TAS3351 on cellular phospho-EGFR was analyzed by In-cell western assay. In addition, inhibition of cell viability in Ba/F3 cells transformed by human wild type or mutated EGFR, as well as in human NSCLC cell lines whose EGFR harbors common mutations, was tested. Anti-tumor activity of TAS3351 was evaluated in NIH/3T3-EGFR allograft models and human NSCLC xenograft models. Results: TAS3351 exhibited almost comparable inhibitory potency against cellular phosphorylation of EGFR harboring ex19del or L858R with or without C797S and/or T790M while sparing wild type EGFR activity. TAS3351 also showed similar trends in the cell viability assay for Ba/F3-EGFR cells and human NSCLC cell lines. Finally, TAS3351 demonstrated favorable anti-tumor activity in NIH/3T3 allografts transformed by human EGFR harboring ex19del/C797S/T790M. Conclusion: TAS3351 is a fourth-generation EGFR-TKI overcoming T790M and C797S-mediated resistance in NSCLC with common mutations. The present results support the clinical development of TAS3351 as monotherapy in NSCLC patients with EGFR mutations harboring C797S and/or T790M EGFR-TKI resistance mutations. Citation Format: Hidefumi Kasuga, Yuki Kataoka, Fuyuki Yamamoto, Takashi Mizutani, Shingo Tsuji, Sakiho Tanaka, Shinji Mizuarai. TAS3351 is a 4th-generation EGFR-TKI overcoming T790M and C797S-mediated resistance in NSCLC with EGFR common mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3259.

MG53 suppresses tumor progression and stress granule formation by modulating G3BP2 activity in non-small cell lung cancer

BackgroundCancer cells develop resistance to chemotherapeutic intervention by excessive formation of stress granules (SGs), which are modulated by an oncogenic protein G3BP2. Selective control of G3BP2/SG signaling is a potential means to treat non-small cell lung cancer (NSCLC).MethodsCo-immunoprecipitation was conducted to identify the interaction of MG53 and G3BP2. Immunohistochemistry and live cell imaging were performed to visualize the subcellular expression or co-localization. We used shRNA to knock-down the expression MG53 or G3BP2 to test the cell migration and colony formation. The expression level of MG53 and G3BP2 in human NSCLC tissues was tested by western blot analysis. The ATO-induced oxidative stress model was used to examine the effect of rhMG53 on SG formation. Moue NSCLC allograft experiments were performed on wild type and transgenic mice with either knockout of MG53, or overexpression of MG53. Human NSCLC xenograft model in mice was used to evaluate the effect of MG53 overexpression on tumorigenesis.ResultsWe show that MG53, a member of the TRIM protein family (TRIM72), modulates G3BP2 activity to control lung cancer progression. Loss of MG53 results in the progressive development of lung cancer in mg53-/- mice. Transgenic mice with sustained elevation of MG53 in the bloodstream demonstrate reduced tumor growth following allograft transplantation of mouse NSCLC cells. Biochemical assay reveals physical interaction between G3BP2 and MG53 through the TRIM domain of MG53. Knockdown of MG53 enhances proliferation and migration of NSCLC cells, whereas reduced tumorigenicity is seen in NSCLC cells with knockdown of G3BP2 expression. The recombinant human MG53 (rhMG53) protein can enter the NSCLC cells to induce nuclear translation of G3BP2 and block arsenic trioxide-induced SG formation. The anti-proliferative effect of rhMG53 on NSCLC cells was abolished with knockout of G3BP2. rhMG53 can enhance sensitivity of NSCLC cells to undergo cell death upon treatment with cisplatin. Tailored induction of MG53 expression in NSCLC cells suppresses lung cancer growth via reduced SG formation in a xenograft model.ConclusionOverall, these findings support the notion that MG53 functions as a tumor suppressor by targeting G3BP2/SG activity in NSCLCs.

Abstract 2423: Discovery of ZN-e4, an irreversible EGFR-TKI with potent anti-tumor activity in EGFR mutant non-small-cell lung cancer

Abstract Small molecule tyrosine kinase inhibitors against the epidermal growth factor (EGFR) have made significant impact in the treatment of advanced non-small-cell lung cancer (NSCLC) with activating EGFR mutations. Although the first and second-generation EGFR inhibitors have remarkably improved survival in advanced EGFR mutant NSCLC patients, most of the patients develop acquired resistance. The gatekeeper mutation (T790M) is the most common acquired resistance mechanism to the first and second-generation EGFR inhibitors. To overcome the T790M acquired resistance, third-generation EGFR inhibitors, such as osimertinib, were developed. However, additional mutant selective EGFR inhibitors with improved toxicity profile are still needed as osimertinib treatment produced incidence of adverse events, such as diarrhea and skin rush. Here, we describe the identification and characterization of ZN-e4, an orally bioavailable, selective, irreversible third generation EGFR inhibitor. ZN-e4 selectively inhibited the kinase activity of several mutated forms of EGFR, including L858R, T790M/L858R, and Exon19 del in biochemical assay. In cell-based assays, ZN-e4 demonstrated a 20-40-fold selectivity ratio for mutant EGFR forms over the wild-type form thus minimizing the potential for on target toxicity. ZN-e4 potently inhibited the proliferation of NSCLC cell lines harboring EGFR activating and T790M mutation and induced tumor regression in small and large tumors with activating EGFR mutations in preclinical human NSCLC xenograft models. Taken together, our data suggest that ZN-e4 is a potent and selective third generation EGFR inhibitor. Currently, ZN-e4 is in a Phase I clinical trial in patients with EGFR mutant NSCLC showing clinical activity and encouraging toxicity profile. Citation Format: Jiali Li, Sunny Abraham, Jianhui Ma, Peter Q. Huang, Kevin D. Bunker, Fernando Doñate, Ahmed A. Samatar. Discovery of ZN-e4, an irreversible EGFR-TKI with potent anti-tumor activity in EGFR mutant non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2423.

Abstract 1874: Anti-VEGF therapy sensitizes EGFR/MET co-altered NSCLC cells to TKIs via inhibiting the VEGF/ERK/MET pathway

Abstract Mesenchymal epithelial transition factor (MET) activation has been considered to mediate primary and acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC), but the mechanisms are not wholly clear. Meanwhile, the effective therapeutic strategy for these patients remains controversial. We previously presented a lung adenocarcinoma patient with dual EGFR-MET alterations benefited from bevacizumab + erlotinib as first-line treatment, achieving 13 months partial response (PR). PFS of another phase reached to 9 months with the combination of osimertinib + crizotinib + bevacizumab when disease proceeded due to increased MET copy number. Persistent PFS benefit was confirmed when the patient was administrated with different TKIs treatment in the presence of bevacizumab. However, the underlying mechanism remains elusive. Thus, we assumed that VEGF might be involved in the regulation of the MET signaling pathway and the addition of anti-VEGF therapy might be promising. In this study, we firstly confirmed whether MET overexpression alone might causally responsible for resistance to gefitinib treatment. Thus, we conducted in vitro forward genetic experiments, we took advantage of the EGFR-mutant lung cancer cell line PC-9, which had been shown to be sensitive to gefitinib. Ectopic overexpression of MET in PC-9 parental cells (PC-9/MET) was able to confer resistance to gefitinib by activating AKT and ERK signaling and sustaining proliferation.Meanwhile we got a gefitinib resistant EGFR mutant NSCLC cell line, HCC827 (HCC827-GR), which showing acquired MET amplification. Interestingly, we found that VEGF promoted cell survival and elicited adaptive resistance to gefitinib and crizotinib alone or in combination in MET transduced PC9-MET cells. This phenomenon also existed in HCC827-GR which harboring MET amplification. Importantly, addition of the VEGF inhibitor bevacizumab was able to restore partial sensitivity to gefitinib in both PC-9/MET and HCC827-GR cells while triple inhibition of EGFR, MET, and VEGF could suppresses the cell proliferation more remarkable. Moreover, we observed that VEGF could facilitate MET phosphorylation and activate several intracellular signaling pathways, including ERK, AKT, and Stat3. Then we pretreated cells with several specific kinase inhibitors, followed by treatment with VEGF. We found that VEGF-mediated up-regulation of MET depended on the ERK pathway and was independent of AKT and Stat3 pathways. These findings were further confirmed in human NSCLC Xenograft models. Addition of bevacizumab to TKIs resulted in stronger inhibition of tumor growth than TKI alone or TKIs combination treatment, by blocking both EGFR and MET signal transduction. In conclusion, bevacizumab + TKIs enhanced antitumor activity in NSCLC with EGFR/MET co-alteration through inhibition of the VEGF/ ERK/MET signaling axis. Citation Format: Qian Chu, Shanshan Huang, Yuan Gao, Jie Lin, Yuan Chen, Bo Zhu, Kongming Wu. Anti-VEGF therapy sensitizes EGFR/MET co-altered NSCLC cells to TKIs via inhibiting the VEGF/ERK/MET pathway [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 1874.

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.

Structure-based design and synthesis of 2,4-diaminopyrimidines as EGFR L858R/T790M selective inhibitors for NSCLC

Mutated epidermal growth factor receptor (EGFR) is a major driver of non-small cell lung cancer (NSCLC). The EGFRT790M secondary mutation has become a leading cause of clinically-acquired resistance to gefitinib and erlotinib. Herein, we present a structure-based design approach to increase the potency and selectivity of the previously reported reversible EGFR inhibitor 7, at the kinase and cellular levels. Three-step structure-activity relationship exploration led to promising compounds 19e and 19h with unique chemical structure and binding mode from the other third-generation tyrosine kinase inhibitors. In a human NSCLC xenograft model, 19e and 19h exhibited dose-dependent tumor growth suppression without toxicity. These selective inhibitors are promising drug candidates for EGFRT790M-driven NSCLC.

Abstract 283: PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances pemetrexed antitumor efficacy in a human nonsquamous NSCLC xenograft model

Abstract Introduction: Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that has been shown to accumulate at high levels in several cancers, including non-small cell lung cancer (NSCLC). The accumulation of HA in NSCLC is associated with a more aggressive phenotype and shortened overall survival. In a prevalence analysis using tissue microarrays of archival human specimens (N = 194) stained by affinity histochemistry, we demonstrated that 55% of adenocarcinomas, 74% of squamous cell carcinomas and 83% of large cell carcinomas accumulated HA. The engineered enzyme PEGylated recombinant human hyaluronidase PH20 (PEGPH20) removes HA from the tumor microenvironment. PEGPH20-based therapies are being evaluated in clinical studies of several solid tumors, including NSCLC (NCT02346370, NCT02563548). Herein, we evaluated the hypothesis that PEGPH20 combination therapy could enhance the efficacy of pemetrexed (PEM), a folate antimetabolite approved by FDA as a first-line treatment in combination with cisplatin, against locally advanced and metastatic NSCLC in patients with non-squamous histology. Methods: We evaluated PEGPH20 plus PEM in a NSCLC xenograft model. First, the human NSCLC adenocarinoma cell line A549 was transduced with hyaluronan synthase-3 (HAS3) to generate the A549/HAS3 cell line. Next, A549/HAS3 cells were inoculated adjacent to the tibial periosteum of nude mice and tumor growth was monitored via ultrasonography. When tumors size reached ∼250 mm3, mice were staged into six groups: (1) vehicle; (2) PEGPH20, (3) PEM daily, (4) PEM weekly, (5) PEGPH20+PEM daily or (6) PEGPH20+PEM weekly. Vascular volume, hypoxia and tumor growth inhibition (TGI) were assessed using conventional methods. Affinity histochemical staining for HA was performed on formalin-fixed paraffin-embedded tissue sections after harvest. Results: A549/HAS3 tumors grew faster than parental A549 tumors in vivo. PEGPH20 monotherapy significantly reduced tumoral HA, increased vascular volume and reduced hypoxia in tumors. Further, TGI with PEGPH20+PEM, weekly or daily, was 29.7 or 31.1%, respectively, whereas in either PEGPH20 alone or PEM alone, both were <9%. Staining of A549/HAS3 tumors showed increased accumulation of HA as compared with the parental A549 xenografts. Conclusion: These data suggest that addition of PEGPH20 to pemetrexed increases the efficacy of PEM and support further investigation of PEGPH20 plus PEM treatment in NSCLC tumors of non-squamous histology that accumulate HA. Citation Format: Renee Clift, Jessica A. Cowell, Susan J. Zimmerman, Mathieu Marella, Ping Jiang, Arnold B. Gelb, Michael J. LaBarre, Daniel C. Maneval, Curtis B. Thompson, Xiaoming Li. PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances pemetrexed antitumor efficacy in a human nonsquamous NSCLC xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 283.

Inhibition of the JAK/STAT pathway with ruxolitinib overcomes cisplatin resistance in non-small-cell lung cancer NSCLC.

This study was aimed to elucidate the roles of inhibition of related JAK/STAT pathways in regulating cytotoxicity induced by cisplatin in non-small-cell lung cancer (NSCLC) cell. We treated five non-small-cell lung cancer cell lines with cisplatin alone or with cisplatin and Jak2 inhibitor (ruxolitinib) and assessed cell viability, expression of Jak2 and STAT3 and cell apoptosis. We also investigated the effect of combination treatment inhibited tumor xenograft growth in two human NSCLC xenograft models bearing the cisplatin resistant (H1299) and sensitive (A549) cells. Different cell lines with different genetic background showed half-maximal inhibitory concentrations (IC50) of cisplatin from 4.66 to 68.28 µmol/L. They could be divided into cisplatin intrinsic resistant and cisplatin sensitive cell lines. In cisplatin-resistant cells with higher Jak2 and STAT3 expression, cisplatin and ruxolitinib combination dramatically suppressed the cell growth, down-regulated the expression of phosphorylated STAT3 and induced cleaved caspase-3 expression. Moreover combination with cisplatin and ruxolitinib also significantly inhibited the growth of resistant cell H1299, A549/DDP and H2347 in soft agar model. Finally, combination group significant inhibited the tumor growth and induced the caspase-3 expression compared with either single agent alone (P < 0.05) on the resistant cell xenografts model. The present study indicates that further study is warranted to determine the effectiveness of combination treatment with cisplatin and Jak2/stat3 pathway inhibitor for platinum-resistant NSCLC.

Abstract 2232: Overexpression of the mu opioid receptor in human non-small cell lung cancer promotes Akt and mTOR activation, tumor growth and metastasis

Abstract Background: Recent epidemiologic studies suggesting that there were differences in cancer recurrence contingent on anesthetic regimens have raised the possibility that mu opioid agonists can influence cancer progression. Based on our previous studies indicating the mu opioid receptor (MOR) is upregulated in several types of non-small cell lung cancer (NSCLC), this study examined the functional significance of MOR overexpression to elucidate a possible mechanism for the epidemiologic findings. Methods: Stable vector control (VC) and MOR1 overexpressing human NSCLC bronchioloalveolar carcinoma (BAC) cells were evaluated using immunoblot analysis, proliferation and transendothelial extravasation assays with or without Akt inhibitor, mTOR inhibitor (temsirolimus) or the peripheral MOR antagonist, methylnaltrexone (MNTX). In human NSCLC xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in nude mouse lungs. Results: We provide evidence that MOR is an important regulator of lung cancer progression, a disease with high mortality and few treatment options. MOR overexpression increased Akt and mTOR activation, proliferation and extravasation in human BAC cells. In vivo, overexpression of MOR in BAC increased primary tumor growth rates in nude mice by ∼2.5 fold and lung metastasis by ∼20 fold compared to VC BAC cells. Conclusions: Our overexpression data suggests a possible direct effect of MOR on Akt and mTOR activation and lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. Our observations further suggest that exploration of MOR in NSCLC merits further study both as a diagnostic and therapeutic option. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2232. doi:1538-7445.AM2012-2232

Abstract B23: Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models

Abstract An increasing body of evidence supports the notion that long noncoding RNAs (lncRNAs) play important roles in diseases including cancer. MALAT1 (also called NEAT2) was originally identified in tumors of highly metastatic non-small cell lung cancer (NSCLC), however, the functional role of MALAT1 in carcinogenesis has remained largely unknown. However, MALAT1 is highly expressed in various types of cancer and is implicated in the regulation of alternative splicing. To determine if MALAT1 constitutes an important driver of tumor growth in vivo, we developed potent antisense oligonucleotides (ASOs) that were able to achieve strong inhibition of MALAT1 in the tumor cells of a variety of preclinical models following systemic delivery. MALAT1 ASOs were well tolerated in rodents at doses leading to &amp;gt;95% inhibition of MALAT1 RNA in the liver. Inhibition of MALAT1 expression in tumor and tumor-associated stromal cells was determined by both q-RT-PCR using species-specific probe/primer sets and/or in situ ‘viewRNA’ technology, where target knockdown can be visualized on a cell by cell basis. Systemic administration of mouse MALAT1 ASOs resulted in a decrease in the numbers of polyps and proliferation index (BrdU (+)) in the small intestine of Apcmin mouse model of colon cancer and correlated well with MALAT1 inhibition in the polyps. Mouse MALAT1 ASOs were also effective in a DEN-induced HCC model, where ASO treatment reduced the target RNA ~ 90% in tumor cells with a concomitant decrease in tumor numbers, while control ASO had no effects on either measure. In addition, higher expression of MALAT1 in non-treated tumors compared to adjacent normal hepatocytes was also clearly visualized by the in situ ‘view RNA’ method. Furthermore, MALAT1 ASO significantly delayed tumor growth in C26 colon cancer and reduced tumor size in TRAMP mouse model of prostate cancer, where the target RNA was decreased by 80% in tumor cells. In a human NSCLC patient-derived xenograft model, both significant MALAT1 RNA reduction and a delay in tumor growth were achieved after MALAT1 ASO treatment. The effects of MALAT1 downregulation by ASO were not limited to the inhibition of tumor growth alone. MALAT1 ASO treatment not only inhibited the growth of primary tumors in the EBC-1 human NSCLC xenograft model, but also resulted in a decrease in lung metastasis as measured by micro CT scanning. Furthermore, cross-species MALAT1 ASOs greatly improved the survival of animals bearing Hep3B human hepatocellular carcinoma (HCC) tumor orthotopically (48.5 days with control ASO vs 88 days with MALAT1 ASO, p=0.005). Taken together, these results demonstrate previously undiscovered roles of MALAT1 as an important regulator in vivo tumor growth and metastasis and suggest that selective inhibition of MALAT1 by ASO could have therapeutic value for the cancer treatment. Citation Format: Jeff Hsu, Guobin He, Gourab Bhattacharjee, Tianyuan Zhou, Chris May, Xiaokun Xiao, Gene Hung, Brett P. Monia, A. Robert MacLeod, Youngsoo Kim. Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B23.

Antitumor activity of motesanib alone and in combination with cisplatin or docetaxel in multiple human non–small-cell lung cancer xenograft models

BackgroundNon–small-cell lung cancer (NSCLC) is categorized into various histologic subtypes that play an important role in prognosis and treatment outcome. We investigated the antitumor activity of motesanib, a selective antagonist of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3, platelet-derived growth factor receptor, and Kit, alone and combined with chemotherapy in five human NSCLC xenograft models (A549, Calu-6, NCI-H358, NCI-H1299, and NCI-H1650) containing diverse genetic mutations.ResultsMotesanib as a single agent dose-dependently inhibited tumor xenograft growth compared with vehicle in all five of the models (P < 0.05). When combined with cisplatin, motesanib significantly inhibited the growth of Calu-6, NCI-H358, and NCI-H1650 tumor xenografts compared with either single agent alone (P < 0.05). Similarly, the combination of motesanib plus docetaxel significantly inhibited the growth of A549 and Calu-6 tumor xenografts compared with either single agent alone (P < 0.05). In NCI-H358 and NCI-H1650 xenografts, motesanib with and without cisplatin significantly decreased tumor blood vessel area (P < 0.05 vs vehicle) as assessed by anti-CD31 staining. Motesanib alone or in combination with chemotherapy had no effect on tumor cell proliferation in vitro.ConclusionsThese data demonstrate that motesanib had antitumor activity against five different human NSCLC xenograft models containing diverse genetic mutations, and that it had enhanced activity when combined with cisplatin or docetaxel. These effects appeared to be mediated primarily by antiangiogenic mechanisms.

YM155, a novel survivin suppressant, enhances taxane-induced apoptosis and tumor regression in a human Calu 6 lung cancer xenograft model

Survivin, an apoptotic inhibitor, is overexpressed in the majority of human tumor types and represents a novel target for anticancer therapy. Taxanes induce a mitotic cell-cycle block through the inhibition of microtubule depolymerization, with subsequent elevated expression/stabilization of survivin. We investigated the administration of survivin suppressant YM155 monobromide (YM155), in combination with docetaxel, in a human non-small-cell lung cancer (NSCLC) xenograft model. Animals received a 7-day continuous infusion of YM155, 2 mg/kg, and/or three bolus doses of docetaxel, 20 mg/kg, according to three dosing schedules: YM155 administered concomitantly with docetaxel, before docetaxel, and after docetaxel. YM155 administered either concomitantly with or before docetaxel showed significant antitumor activity (tumor regression ≥ 99%), with complete regression of the established human NSCLC-derived tumors in mice (eight of eight and seven of eight animals, respectively). Significantly fewer complete responses (three of eight animals) were achieved when YM155 was administered after docetaxel. No statistically significant decreases in body weight were observed in the combination versus docetaxel groups. YM155 administered concomitantly with docetaxel resulted in significant decreases in mitotic and proliferative indices, and in a significant increase in the apoptosis index. Elevated survivin expression was seen in tumors from mice treated with docetaxel alone; a significant reduction in survivin expression was seen in tumors from mice treated with YM155 alone or in combination with docetaxel, but not in the control group. These results indicate that in a human NSCLC xenograft model YM155 in combination with docetaxel diminished the accumulation of survivin by docetaxel and induced more intense apoptosis and enhanced antitumor activity, compared with single-agent YM155 or docetaxel.

Paclitaxel Efficacy is Increased by Parthenolide via Nuclear Factor- KappaB Pathways in In Vitro and In Vivo Human Non – Small Cell Lung Cancer Models

The focus of this study was to develop additive or synergistic agents to chemosensitize the existing chemotherapeutic drug in human non-small cell lung cancer (NSCLC). In this study employing analyses of the NF-κB/ I-κB kinase (IKK) signal cascade in a number of NSCLC cell lines, we report the identification and characterization of parthenolide. Parthenolide is a sesquiterpene lactone that can antagonize paclitaxel-mediated NF-κB nuclear translocation and activation through selectively targeting I-κB kinase (IKK) activity. Our results showed that parthenolide dramatically lowered the effective dose of Paclitaxel needed to induce cytotoxicity of a wide range of NSCLC cell lines. An examination of pathways common to Paclitaxel and parthenolide signaling revealed that this synergy was related to modulation of the NF-κB/ I-κB kinase (IKK) signal cascade through IKKβ. Parthenolide alone induced apoptosis via the mitochondria/caspase pathway. Moreover, in a human orthotopic NSCLC xenograft model, a well-tolerated combination induces tumor regression. These data strengthen the rationale for the use of parthenolide to decrease the apoptotic threshold via a caspase-dependent process and support the use of concurrent low doses of paclitaxel in the treatment of NSCLC with paclitaxel chemoresistance.