[Show abstract][Hide abstract] ABSTRACT: The paradigm shift in cancer treatment from cytotoxic drugs to tumor targeted therapies poses new challenges, including optimization of dose and schedule based on a biologically effective dose, rather than the historical maximum tolerated dose. Optimal dosing is currently determined using concentrations of tyrosine kinase inhibitors in plasma as a surrogate for tumor concentrations. To examine this plasma-tumor relationship, we explored the association between lapatinib levels in tumor and plasma in mice and humans, and those effects on phosphorylation of human epidermal growth factor receptors (HER) in human tumors.
[Show abstract][Hide abstract] ABSTRACT: Inflammatory breast Cancer (IBC) is a rare aggressive breast cancer in which cancer cells block the lymph vessels in the skin of the breast. IBC tumors have a high rate of ERBB2 positivity and response to ERBB2 targeted therapies. Due to the rarity of this cancer, the molecular etiology of this disease is poorly understood.
Materials & Methods
A 208 gene next-generation sequencing (NGS) panel was used to analyze 20 IBC patient tumor and matched normal samples. Tissues were stained for cell signaling proteins and immune-markers. Cell line studies were performed to understand the impact of genomic variants on therapeutic selection.
Common pathway alterations reoccur among IBC samples that influence genome stability, PI3K signaling, and ERBB signaling. NGS revealed alterations in both the ERBB/PI3K pathways, including: ERBB2 amplification, ERBB3 mutations, and activating PI3K mutations. Immunohistochemistry (IHC) staining for pS6 and pERBB3 identified ERBB/PI3K pathway activity in IBC samples. Cell line studies using siRNA and neutralizing antibodies demonstrate that mutant ERBB3 signaling contributes to IBC proliferation.
IBC tumors with high levels of CD8+ immune infiltrate have a significantly higher somatic mutation rate than other IBC tumors. A proposed score (iScore) based on the somatic mutation rate and the average mutant allele frequency, showed greater correlation with the level of CD8+ immune infiltration. Furthermore, tumors with high CD8+ infiltrating lymphocytes were associated with a higher frequency of alterations in DNA mismatch repair (MMR) genes. IHC revealed high levels of the immune checkpoint signaling molecule PD-L1 in the inflammatory infiltrate of IBC tumors.
This study identifies a higher level of ERBB3 mutations than reported in other cancers and an important role for ERBB3 mutation in IBC. ERBB3 targeted therapies in combination with ERBB/PI3K drugs may be important for IBC treatment. Furthermore, high somatic mutations rates in a subset of IBC tumors harboring MMR mutations lead to greater levels of PD-L1+ immune infiltrates, which suggests a possible benefit from immunotherapies such as anti-PD-L1 antibodies. iScore, a more predictive value of immune infiltration in tumors, may be indicative of the level of neoantigen exposure to the immune system. The utility of the newly proposed iScore is currently being investigated as a method to predict immune cell infiltrates and immunotherapy response in other tumor types.
[Show abstract][Hide abstract] ABSTRACT: Ponatinib, a multi-targeted TKI and potent pan-ABL inhibitor, approved for the treatment of Ph+ALL and CML, was temporarily withdrawn from the US market due to severe vascular adverse events. Cardiac-specific toxicities including myocardial infarction, severe congestive heart failure, and cardiac arrhythmias have also been shown with ponatinib. Targeted oncology agents such as ponatinib have transformed cancer treatment but often induce toxicity due to inhibition of survival pathways shared by both cancer and cardiac cells. These toxicities are often missed by the standard preclinical toxicity assessment methods, which include human Ether-à-go-go-Related Gene (hERG) and animal toxicity testing. In this study, we show that a multi-parameter in vitro toxicity screening approach using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM)accurately predicted the cardiac toxicity potential of ponatinib. This in vitro model evaluated ponatinib's effect on the overall cell health, mitochondrial stress, and function of hiPSC-CM and also provided mechanistic insight into the signaling pathways and cellular structures altered with treatment. We show here that ponatinib rapidly inhibits pro-survival signaling pathways, induces structural cardiac toxicity (as shown by actin cytoskeleton damage, mitochondrial stress, cell death, and troponin secretion), and disrupts cardiac cell beating. Most of these effects occurred at doses between 10 - 50X ponatinib's Cmax, a dose range shown to be relevant for accurate prediction of in vivo toxicity. Together these studies show that a comprehensive in vitro screening tool in a more relevant human cardiac cell model can improve the detection of cardiac toxicity with targeted oncology agents such as ponatinib.
No preview · Article · Oct 2014 · Toxicological Sciences
[Show abstract][Hide abstract] ABSTRACT: KRAS gene mutation is linked to poor prognosis and resistance to therapeutics in Non Small Cell Lung Cancer (NSCLC). In this study, we have explored the possibility of exploiting inherent differences in KRAS mutant cell metabolism for treatment. This study identified a greater dependency on folate metabolism pathways in KRAS mutant compared to KRAS wild type NSCLC cell lines. Microarray gene expression and biological pathway analysis identified higher expression of folate metabolism and purine synthesis related pathways in KRAS mutant NSCLC cells compared to wildtype counterparts. Moreover, pathway analysis and knockdown studies suggest a role for MYC transcriptional activity in the expression of these pathways in KRAS mutant NSCLC cells. Furthermore, KRAS knockdown and overexpression studies demonstrated the ability of KRAS to regulate expression of genes that comprise folate metabolism pathways. Proliferation studies demonstrated higher responsiveness to methotrexate, pemetrexed and other antifolates in KRAS mutant NSCLC cells. Surprisingly, KRAS gene expression is downregulated in KRAS wildtype and KRAS mutant cells by antifolates which may also contribute to higher efficacy of antifolates in KRAS mutant NSCLC cells. In vivo analysis of multiple tumorgraft models in nude mice identified a KRAS mutant tumor among the pemetrexed responsive tumors and also demonstrated an association between expression of folate pathway gene, Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2), and antifolate activity. Collectively, we identify altered regulation of folate metabolism in KRAS mutant NSCLC cells that may account for higher antifolate activity in this subtype of NSCLC.
Preview · Article · Mar 2014 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] ABSTRACT: The human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2 addicted tumors. While lapatinib, an FDA-approved small molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance.
Using a functional protein pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and other TKIs in class.
The signaling analysis revealed that while HER2 was appropriately inhibited in lapatinib resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR persistent activation, we found that lapatinib resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling as seen in the parental lapatinib sensitive cell lines, but instead were dependent upon an heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3 mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings.
Molecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.
Full-text · Article · Sep 2013 · Breast cancer research: BCR
[Show abstract][Hide abstract] ABSTRACT: Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/ MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development.
No preview · Article · May 2013 · Toxicology and Applied Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor (EGF)-like growth factors control tumor progression as well as evasion from the toxic effects of chemotherapy. Accordingly, antibodies targeting the cognate receptors, such as EGFR/ErbB-1 and the co-receptor HER2/ErbB-2, are widely used to treat cancer patients, but agents that target the EGF-like growth factors are not available. To circumvent the existence of 11 distinct ErbB ligands, we constructed a soluble fusion protein (hereinafter: TRAP-Fc) comprising truncated extracellular domains of EGFR/ErbB-1 and ErbB-4. The recombinant TRAP-Fc retained high-affinity ligand binding to EGF-like growth factors and partially inhibited growth of a variety of cultured tumor cells. Consistently, TRAP-Fc displayed an inhibitory effect in xenograft models of human cancer, as well as synergy with chemotherapy. Additionally, TRAP-Fc inhibited invasive growth of mammary tumor cells and reduced their metastatic seeding in the lungs of animals. Taken together, the activities displayed by TRAP-Fc reinforce critical roles of EGF-like growth factors in tumor progression, and they warrant further tests of TRAP-Fc in preclinical models.
[Show abstract][Hide abstract] ABSTRACT: A phase II study of dasatinib, an inhibitor of multiple oncogenic tyrosine kinases including Src, was conducted to evaluate 16-week progression-free rate and tolerability in patients with previously treated metastatic breast cancer (MBC). Real-time assessment of potential tissue biomarkers of Src inhibition was used to optimize dosing.
Eligibility criteria required that patients have measurable MBC, biopsiable tumor, and unlimited prior therapies. For the analysis of change in protein biomarkers of Src inhibition, focal adhesion kinase, paxillin, and p-Src, patients underwent metastatic biopsies at baseline and 4 weeks. Patients who tolerated the starting dose of dasatinib (50 or 70 mg orally twice daily) for the first 28-day cycle, and displayed suboptimal Src inhibition, were escalated to a higher dose (70 or 100 mg).
The trial was closed early with 31 patients because of a statistical boundary that required at least 4 (13%) patients without disease progression to continue accrual. These 31 patients had a median of 2 prior lines of chemotherapy for MBC. The most notable toxicity was pleural effusions in 16 patients (52%). Twenty patients had evaluable metastatic biopsies. None of the tumors showed the predefined optimal level of Src inhibition at week 4.
Single-agent dasatinib did not exhibit significant antitumor activity in patients with heavily pretreated MBC. There were no clinically meaningful decreases before and after dasatinib exposure between exploratory tissue biomarkers of Src inhibition which may be attributable to challenges in defining biomarker endpoints for multitargeted tyrosine kinase inhibitors.
Preview · Article · Aug 2011 · Clinical Cancer Research