Abstract Alternative splicing (AS) is a primary mechanism for mRNA transcript diversification and protein expression regulation. In cancer, altered mRNA splicing promotes oncogenic transformation, ...induces metastasis, and confers resistance to cancer treatment. Mutations or imbalanced expression/activity of splicing factors (SF), such as serine-arginine-rich SFs (SRSFs), often result in deregulation of RNA splicing and tumor progression. CDC2-like kinases (CLK) and dual-specificity tyrosine-regulated kinase (DYRK) are key regulators of AS via phosphorylation of SRSFs. BH-30236 is a novel macrocyclic ATP-competitive inhibitor of CLK1/2/4. It also inhibits DYRK1/2, provirus integration site for moloney leukemia virus 3 (PIM3) and FMS-like tyrosine kinase 3 (FLT3) at clinically relevant concentrations. The inhibitory activity of BH-30236 against CLK, DYRK, PIM3 and FLT3 in cancer cells was investigated via the phosphorylation inhibition of SRSFs, Tau, 4EBP1 and FLT3, respectively. A panel of 119 hematological and solid tumor cell lines were used to investigate the spectrum of BH-30236 against cancer cell growth. To understand the mechanism of anti-cancer cell growth, the effect of BH-30236 on AS and protein expression of key tumor related biomarkers were examined. In conclusion, BH-30236 effectively inhibited pSRSF (IC50 40-60 nM in IMR-32 cells), pTau (IC50 ~50 nM in SH-SY5Y cells), p4EBP1 (IC50 ~80 nM in MM1S cells) and pFLT3-ITD (IC50 0.16 nM in MV-4-11 cells). BH-30236 demonstrated broad inhibition of cancer cell growth with a median IC50 of 85.2 nM (range 0.55-393 nM) across 12 tumor types. BH-30236 showed great efficacy against cell lines derived from hematological malignancy (median IC50 23.34 nM), neuroblastoma (median IC50 25.73 nM), breast cancer (median IC50 83.80 nM), colon cancer (median IC50 85.17 nM), and lung cancer (median IC50 102.65 nM). Furthermore, BH-30236 demonstrated synergy with KRAS inhibitors in KRAS mutant cell lines, with EGFR inhibitors in RBM10 mutant H1975 cells, and with BCL2 inhibitor Venetoclax in MOLM-13 cells. Mechanistically, BH-30236 modulated AS by increasing pro-apoptotic and anti-proliferative splicing variants of key factors, such as BCL2L1, S6K, and BCLAF1. Meanwhile, BH-30236 downregulated RNA expression of SRSFs and modulated BCL2 family to increase apoptosis. These results strongly support the clinical applications of the novel multikinase CLK inhibitor BH-30236 in hematological malignancies and solid tumors as a single agent or in combination with other therapies. Citation Format: Ping Jiang, Danan Li, Nancy Ling, Dayong Zhai, Wei Deng, Eugene Rui, J. Jean Cui. BH-30236, a novel macrocyclic CLK inhibitor modulating RNA splicing, demonstrates potent inhibition of cancer cell growth in a broad panel of cancer cell lines abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 617.
The discovery of somatic mutations in EGFR and development of EGFR tyrosine kinase inhibitors (TKI) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all ...patients and currently no effective treatment is available. Here, we show that β-catenin is essential for development of EGFR-mutated lung cancers. β-Catenin was upregulated and activated in EGFR-mutated cells. Mutant EGFR preferentially bound to and tyrosine phosphorylated β-catenin, leading to an increase in β-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacologic inhibition of β-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth, and genetic deletion of the β-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that β-catenin plays an essential role in lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs.
Mutations in the HER2 kinase domain have been identified in human clinical lung cancer specimens. Here we demonstrate that inducible expression of the most common HER2 mutant (HER2YVMA) in mouse lung ...epithelium causes invasive adenosquamous carcinomas restricted to proximal and distal bronchioles. Continuous expression of HER2YVMA is essential for tumor maintenance, suggesting a key role for HER2 in lung adenosquamous tumorigenesis. Preclinical studies assessing the in vivo effect of erlotinib, trastuzumab, BIBW2992, and/or rapamycin on HER2YVMA transgenic mice or H1781 xenografts with documented tumor burden revealed that the combination of BIBW2992 and rapamycin is the most effective treatment paradigm causing significant tumor shrinkage. Immunohistochemical analysis of lung tumors treated with BIBW2992 and rapamycin combination revealed decreased phosphorylation levels for proteins in both upstream and downstream arms of MAPK and Akt/mTOR signaling axes, indicating inhibition of these pathways. Based on these findings, clinical testing of the BIBW2992/rapamycin combination in non-small cell lung cancer patients with tumors expressing HER2 mutations is warranted.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Abstract Aberrant alternative splicing is a newly recognized hallmark of cancer, that has been shown to play a critical role in tumorigenesis, cancer progression, and therapeutic resistance via ...multiple mechanisms, including increased proliferation, decreased apoptosis, promoted migration, enhanced metastatic potential, and induced evasion of immune surveillance. Serine and arginine-rich splicing factors (SRSFs) are RNA-binding proteins (RBPs) that regulate constitutive and alternative splicing. SRSFs are often mutated or overexpressed in cancers, resulting in widespread alterations in splicing patterns. The Cdc2-like kinase (CLK) family and dual-specificity tyrosine-regulated kinase (DYRK) phosphorylate SRSFs, influencing the assembly of spliceosome machinery, exon recognition, and splicing. Therefore, targeting CLK/DYRK kinases can modulate cancer specific splicing isoforms, opening avenues for new therapeutic interventions. BH-30236 was designed as a novel orally bioavailable, ATP-competitive, macrocyclic inhibitor of CLK with IC50s of 0.134, 0.165, and 0.446 nM against CLK1, CLK2, and CLK4, respectively in enzymatic kinase assays. At clinically relevant concentrations, BH-30236 also inhibited DYRK1A/1B/2, proviral insertion site of moloney murine leukemia virus kinase 3 (PIM3), and FMS-like tyrosine kinase 3 (FLT3) with IC50s of 0.111, 0.148, 0.562, 0.115 and 0.248 nM, respectively. In cancer cells, BH-30236 impaired the phosphorylation of SRSFs, Tau and 4EBP1, the direct downstream substrates of CLK, DYRK, and PIM kinases with IC50s of 40-60 nM, ~50 nM, and ~80 nM, respectively. Furthermore, BH-30236 potently inhibited the FLT3 phosphorylation with an IC50 of 0.16 nM. Overall, BH-30236 regulated alternative splicing by primarily inducing skipped exons in favor of anti-tumor isoforms, leading to cancer cell death and growth suppression in a broad panel of cancer cell lines and in vivo efficacy studies. For example, BH-30236 potently inhibited cell proliferation with an IC50 of 0.62 nM in FLT3-ITD positive MV-4-11 cells and achieved complete tumor regression in the MV-4-11 cell-derived xenograft tumor model, even after dose cessation for 4 weeks. In MV-4-11 cells, BH-30236 increased pro-apoptosis splicing variant BCLxS, downregulated RNA expression of BCL2, MCL1, and AML stem cell markers CD33 and CD123. In addition, BH-30236 has demonstrated good human ADME and preclinical safety profiles. Collectively, the preclinical study results strongly support the clinical applications of the novel multikinase CLK inhibitor BH-30236 in hematological malignancies and solid tumors, as a single agent or in combination with other therapies. Citation Format: J. Jean Cui, Ping Jiang, Wei Deng, Dayong Zhai, Nancy Ling, Danan Li, Zhenping Wang, Yue Hu, Levan Darjania, Jeff Whitten, Evan Rogers, Eugene Rui. Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5944.
Abstract
While the Notch pathway is reportedly activated in breast cancer, the molecular mechanisms leading to its aberrant activation remain elusive, hampering the optimal development of Notch ...inhibitors in the clinics. In an effort to identify predictive biomarkers of response to Notch targeted therapies in breast cancer, we used several computational approaches, including novel algorithms for detecting complex structural rearrangements, to analyze next generation sequencing and related omic datasets from The Cancer Genome Atlas (TCGA) breast cancer cohort. We identified simple mutations and complex rearrangements in NOTCH1, NOTCH2 and NOTCH3 that compromised the function of the PEST domain, a negative regulatory domain controlling the duration of active Notch signaling. Focal amplifications of NOTCH2 and NOTCH3 were also observed, as were heterodimerization or extracellular domain alterations, at lower incidence. Mutations and amplifications often activated the Notch pathway as evidenced by increased expression of canonical Notch target genes, and functional mutations were significantly enriched in the triple negative breast cancer (TNBC) subtype. We also sequenced a panel of breast cancer xenograft models and identified models that harbor functionally equivalent PEST domain alterations. These models were significantly more sensitive to a gamma secretase inhibitor (GSI) compared to models without Notch alterations. Gene expression and functional analyses were performed to study the mechanism of activation through mutation and inhibition by GSI. In summary, we demonstrated that Notch pathway is activated via multiple mutational mechanisms primarily involving the PEST domains of NOTCH1, NOTCH2 and NOTCH3. Collectively, approximately 13% of TNBC exhibits a genetic alteration coupled with pathway up-regulation and these alterations may serve as biomarkers to identify patients most likely to respond to Notch inhibitors.
Citation Format: Kai Wang, Qin Zhang, Danan Li, Keith Ching, Cathy Zhang, Xianxian Zheng, Mark Ozeck, Stephanie Shi, Xiaorong Li, Hui Wang, Paul Rejto, James Christensen, Peter Olson. Identification of drug-sensitive Notch receptor alterations in human breast cancer. abstract. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-52.
Germline TSC1 or TSC2 mutations cause tuberous sclerosis complex (TSC), a hamartoma syndrome with lung involvement. To explore the potential interaction between TSC1 and KRAS activation in lung ...cancer, mice in which Tsc1 loss and Kras(G12D) expression occur in a small fraction of lung epithelial cells were generated. Mice with a combined Tsc1-Kras(G12D) mutation had dramatically reduced tumor latency (median survival: 11.6-15.6 weeks) in comparison with Kras(G12D) alone mutant mice (median survival: 27.5 weeks). Tsc1-Kras(G12D) tumors showed consistent activation of mTOR (mammalian target of rapamycin)C1 and responded to treatment with rapamycin, leading to significantly improved survival, whereas rapamycin had minor effects on cancers in Kras(G12D) alone mice. Loss of heterozygosity for TSC1 or TSC2 was found in 22% of 86 human lung cancer specimens. However, none of the 80 lung cancer lines studied showed evidence of the lack of expression of either TSC1 or TSC2 or a signaling pattern corresponding to complete loss. These data indicate that Tsc1 loss synergizes with the Kras mutation to enhance lung tumorigenesis in the mouse, but that this is a rare event in human lung cancer. Rapamycin may have unique benefit for patients with lung cancer, for whom the TSC1/TSC2 function is limited.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract
The discovery of somatic mutations in epidermal growth factor receptor (EGFR) and the development of EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have ...revolutionized treatment for non-small cell lung cancer (NSCLC). Resistance to TKIs emerges in almost all patients, but currently no effective treatment is available.Therefore, novel strategies to either prevent or overcome resistance are sorely needed. Here we show that β-catenin is essential for development of EGFR mutated lung cancers. We found that β-catenin was upregulated, translocated to the nucleus, and subsequently activated in both EGFR mutated lung cancer cell lines and EGFR mutation driven lung tumors. We demonstrated that mutant EGFR preferentially bound to β-catenin and caused tyrosine-phosphorylation of β-catenin. Tyrosine-phosphorylation of β-catenin led to stabilization, nuclear translocation, and transcriptional activity particularly in cells harboring EGFR-L858R (LR)-T790M (TM). Pharmacological β-catenin inhibition using ICG-001, which specifically blocks the CBP-β-catenin interaction, suppressed growth of both H1975 cells harboring EGFR-LR-TM and lung tumors in EGFR-LR-TM transgenic mice. To further examine whether β-catenin plays an important role in lung tumorigenesis, we generated an EGFR-LR-TM lung cancer mouse model in which the β-catenin gene (Ctnnb1) can be conditionally deleted. Genetic deletion of Ctnnb1 dramatically reduced lung tumor formation and showed significantly longer survival. Taken together, our data suggest that β-catenin plays an important role in mutant EGFR-driven lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development and/or overcome resistance to EGFR TKIs.
Citation Format: Sohei Nakayama, Natasha J. Sng, Julian Carretero, Robert Welner, Yuichiro Hayashi, Mihoko Yamamoto, Tan J. Alistair, Norihiro Yamaguchi, Hiroyuki Yasuda, Li Danan, Kenzo Soejima, Soo A. Ross, Costa B. Daniel, Kwok-Kin Wong, Susumu S. Kobayashi. β-catenin plays an important role in lung tumor development induced by EGFR mutations. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 968. doi:10.1158/1538-7445.AM2014-968
The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced ...microCT (μCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially
available μCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal
increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures,
and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. μCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4-ALK mice with crizotinib yielded
65% reduction in tumor size and thus demonstrated the utility of quantitative μCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels
and lung tumors. Contrast-enhanced μCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.
Abstract
Dual HER2 and EGFR inhibitor BIBW2992 have dramatic activity in a HER2 wild type/EGFR L858R murine lung cancer model
HER2 (ERBB2) is a proto-oncogene that belongs to EGFR tyrosine kinase ...receptor family. HER2 genomic locus amplification and HER2 protein over-expression detected by IHC have been identified in many human cancer types including breast, brain and lung cancer. Simultaneous over-expression of both HER2 and EGFR occurs in a subpopulation of lung cancer patients. Here we developed a transgenic mouse model with inducible wild type HER2 expression in type II pneumocyte. Continual HER2 over-expression in the lung epithelial compartment leads to peripheral lung adenocarcinoma that responds dramatically to targeted anti-HER2 therapeutics. Mice over-expressing both EGFR L858R and the wild type HER2 alleles do not significant accelerate tumorigenesis or alter of tumor histology as compared to tumors generated by the wild type HER2 single alleles. The HER2/EGFR driven murine lung tumors showed mild and transient response to the EGFR inhibitor, gefitinib. This result mimics the observation from clinical patients. However, the dual HER2/EGFR inhibitor, BIBW2992, is much more effective against these HER2/EGFR driven lung cancers as measured by tumor regression and over all survival. Our data suggested that targeting therapy against both EGFR and HER2 is a better strategy in lung cancer patients with co-expression of EGFR and HER2 when compared with treatment against EGFR alone. Clinical trial for dual HER2/EGFR inhibitors in this population should be considered.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3228.
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