Danan Li

BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.

A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene

Oncogenic mutations in the small GTPase Ras are highly prevalent in cancer, but an understanding of the vulnerabilities of these cancers is lacking. We undertook a genome-wide RNAi screen to identify synthetic lethal interactions with the KRAS oncogene. We discovered a diverse set of proteins whose depletion selectively impaired the viability of Ras mutant cells. Among these we observed a strong enrichment for genes with mitotic functions. We describe a pathway involving the mitotic kinase PLK1, the anaphase-promoting complex/cyclosome, and the proteasome that, when inhibited, results in prometaphase accumulation and the subsequent death of Ras mutant cells. Gene expression analysis indicates that reduced expression of genes in this pathway correlates with increased survival of patients bearing tumors with a Ras transcriptional signature. Our results suggest a previously underappreciated role for Ras in mitotic progression and demonstrate a pharmacologically tractable pathway for the potential treatment of cancers harboring Ras mutations.

LKB1 modulates lung cancer differentiation and metastasis.

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz–Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.

Differential induction of apoptosis in HER2 and EGFR addicted cancers following PI3K inhibition

Non-small cell lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Such cancers are “addicted” to EGFR, and treatment with a TKI invariably leads to down-regulation of the PI3K-AKT-mTOR and MEK-ERK signaling pathways, resulting in apoptosis. Using a dual PI3K-mTOR inhibitor, NVP-BEZ235, we evaluated whether PI3K-mTOR inhibition alone induced apoptosis in these cancers. In contrast to HER2-amplified breast cancers, we found that PI3K-mTOR inhibition did not promote substantial apoptosis in the EGFR mutant lung cancers. However, blocking both PI3K-mTOR and MEK simultaneously led to apoptosis to similar levels as the EGFR TKIs, suggesting that down-regulation of these pathways may account for much of the apoptosis promoted by EGFR inhibition. In EGFR mutant lung cancers, down-regulation of both intracellular pathways converged on the BH3 family of proteins regulating apoptosis. PI3K inhibition led to down-regulation of Mcl-1, and MEK inhibition led to up-regulation of BIM. In fact, down-regulation of Mcl-1 by siRNA was sufficient to sensitize these cancers to single-agent MEK inhibitors. Surprisingly, an AKT inhibitor did not decrease Mcl-1 levels, and when combined with MEK inhibitors, failed to induce apoptosis. Importantly, we observed that the combination of PI3K-mTOR and MEK inhibitors effectively shrunk tumors in a transgenic and xenograft model of EGFR T790M-L858R cancers. These data indicate simultaneous inhibition of PI3K-mTOR and MEK signaling is an effective strategy for treating EGFR mutant lung cancers, including those with acquired resistance to EGFR TKIs.

An ErbB3 Antibody, MM-121, Is Active in Cancers with Ligand-Dependent Activation

ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in epidermal growth factor receptor (EGFR; ErbB1), ErbB2 [human epidermal growth factor receptor 2 (HER2)], and [hepatocyte growth factor receptor (MET)] addicted cancers, and reactivation of ErbB3 is a prominent method for cancers to become resistant to ErbB inhibitors. In this study, we evaluated the in vivo efficacy of a therapeutic anti-ErbB3 antibody, MM-121. We found that MM-121 effectively blocked ligand-dependent activation of ErbB3 induced by either EGFR, HER2, or MET. Assessment of several cancer cell lines revealed that MM-121 reduced basal ErbB3 phosphorylation most effectively in cancers possessing ligand-dependent activation of ErbB3. In those cancers, MM-121 treatment led to decreased ErbB3 phosphorylation and, in some instances, decreased ErbB3 expression. The efficacy of single-agent MM-121 was also examined in xenograft models. A machine learning algorithm found that MM-121 was most effective against xenografts with evidence of ligand-dependent activation of ErbB3. We subsequently investigated whether MM-121 treatment could abrogate resistance to anti-EGFR therapies by preventing reactivation of ErbB3. We observed that an EGFR mutant lung cancer cell line (HCC827), made resistant to gefitinib by exogenous heregulin, was resensitized by MM-121. In addition, we found that a de novo lung cancer mouse model induced by EGFR T790M-L858R rapidly became resistant to cetuximab. Resistance was associated with an increase in heregulin expression and ErbB3 activation. However, concomitant cetuximab treatment with MM-121 blocked reactivation of ErbB3 and resulted in a sustained and durable response. Thus, these results suggest that targeting ErbB3 with MM-121 can be an effective therapeutic strategy for cancers with ligand-dependent activation of ErbB3.

Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors.

The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2–7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation.

Predicting drug susceptibility of non–small cell lung cancers based on genetic lesions

Somatic genetic alterations in cancers have been linked with response to targeted therapeutics by creation of specific dependency on activated oncogenic signaling pathways. However, no tools currently exist to systematically connect such genetic lesions to therapeutic vulnerability. We have therefore developed a genomics approach to identify lesions associated with therapeutically relevant oncogene dependency. Using integrated genomic profiling, we have demonstrated that the genomes of a large panel of human non-small cell lung cancer (NSCLC) cell lines are highly representative of those of primary NSCLC tumors. Using cell-based compound screening coupled with diverse computational approaches to integrate orthogonal genomic and biochemical data sets, we identified molecular and genomic predictors of therapeutic response to clinically relevant compounds. Using this approach, we showed that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations confer enhanced Hsp90 dependency and validated this finding in mice with KRAS-driven lung adenocarcinoma, as these mice exhibited dramatic tumor regression when treated with an Hsp90 inhibitor. In addition, we found that cells with copy number enhancement of v-abl Abelson murine leukemia viral oncogene homolog 2 (ABL2) and ephrin receptor kinase and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) kinase family genes were exquisitely sensitive to treatment with the SRC/ABL inhibitor dasatinib, both in vitro and when it xenografted into mice. Thus, genomically annotated cell-line collections may help translate cancer genomics information into clinical practice by defining critical pathway dependencies amenable to therapeutic inhibition.

Exploiting cancer cell vulnerabilities to develop a combination therapy for ras-driven tumors.

Ras-driven tumors are often refractory to conventional therapies. Here we identify a promising targeted therapeutic strategy for two Ras-driven cancers: Nf1-deficient malignancies and Kras/p53 mutant lung cancer. We show that agents that enhance proteotoxic stress, including the HSP90 inhibitor IPI-504, induce tumor regression in aggressive mouse models, but only when combined with rapamycin. These agents synergize by promoting irresolvable ER stress, resulting in catastrophic ER and mitochondrial damage. This process is fueled by oxidative stress, which is caused by IPI-504-dependent production of reactive oxygen species, and the rapamycin-dependent suppression of glutathione, an important endogenous antioxidant. Notably, the mechanism by which these agents cooperate reveals a therapeutic paradigm that can be expanded to develop additional combinations.

Inhibition of ALK, PI3K/MEK, and HSP90 in Murine Lung Adenocarcinoma Induced by EML4-ALK Fusion Oncogene

Genetic rearrangements of the anaplastic lymphoma kinase (ALK) kinase occur in 3% to 13% of non-small cell lung cancer patients and rarely coexist with KRASor EGFR mutations. To evaluate potential treatment strategies for lung cancers driven by an activated EML4-ALK chimeric oncogene, we generated a genetically engineered mouse model that phenocopies the human disease where this rearranged gene arises. In this model, the ALK kinase inhibitor TAE684 produced greater tumor regression and improved overall survival compared with carboplatin and paclitaxel, representing clinical standard of care. 18F-FDG-PET-CT scans revealed almost complete inhibition of tumor metabolic activity within 24 hours of TAE684 exposure. In contrast, combined inhibition of the PI3K/AKT and MEK/ERK1/2 pathways did not result in significant tumor regression. We identified EML4-ALK in complex with multiple cellular chaperones including HSP90. In support of a functional reliance, treatment with geldanamycin-based HSP90 inhibitors resulted in rapid degradation of EML4-ALK in vitro and substantial, albeit transient, tumor regression in vivo. Taken together, our findings define a murine model that offers a reliable platform for the preclinical comparison of combinatorial treatment approaches for lung cancer characterized by ALK rearrangement.