Identification of a Synthetic Lethal Relationship between Nucleotide Excision Repair Deficiency and Irofulven Sensitivity in Urothelial Cancer.

PURPOSE: Cisplatin-based chemotherapy is a first-line treatment for muscle-invasive and metastatic urothelial cancer. Approximately 10% of bladder urothelial tumors have a somatic missense mutation in the nucleotide excision repair (NER) gene, ERCC2, which confers increased sensitivity to cisplatin-based chemotherapy. However, a significant subset of patients is ineligible to receive cisplatin-based therapy due to medical contraindications, and no NER-targeted approaches are available for platinum-ineligible or platinum-refractory ERCC2-mutant cases. EXPERIMENTAL DESIGN: We used a series of NER-proficient and NER-deficient preclinical tumor models to test sensitivity to irofulven, an abandoned anticancer agent. In addition, we used available clinical and sequencing data from multiple urothelial tumor cohorts to develop and validate a composite mutational signature of ERCC2 deficiency and cisplatin sensitivity. RESULTS: We identified a novel synthetic lethal relationship between tumor NER deficiency and sensitivity to irofulven. Irofulven specifically targets cells with inactivation of the transcription-coupled NER (TC-NER) pathway and leads to robust responses in vitro and in vivo, including in models with acquired cisplatin resistance, while having minimal effect on cells with intact NER. We also found that a composite mutational signature of ERCC2 deficiency was strongly associated with cisplatin response in patients and was also associated with cisplatin and irofulven sensitivity in preclinical models. CONCLUSIONS: Tumor NER deficiency confers sensitivity to irofulven, a previously abandoned anticancer agent, with minimal activity in NER-proficient cells. A composite mutational signature of NER deficiency may be useful in identifying patients likely to respond to NER-targeting agents, including cisplatin and irofulven.See related commentary by Jiang and Greenberg, p. 1833.

Leveraging Systematic Functional Analysis to Benchmark an In Silico Framework Distinguishes Driver from Passenger MEK Mutants in Cancer.

Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on MAP2K1 and MAP2K2 mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part in silico methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. In silico prediction accurately distinguished functional from benign MAP2K1 and MAP2K2 mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted in silico modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response. SIGNIFICANCE: Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation.See related commentary by Whitehead and Sebolt-Leopold, p. 4042.

Modeling biological and genetic diversity in upper tract urothelial carcinoma with patient derived xenografts.

Treatment paradigms for patients with upper tract urothelial carcinoma (UTUC) are typically extrapolated from studies of bladder cancer despite their distinct clinical and molecular characteristics. The advancement of UTUC research is hampered by the lack of disease-specific models. Here, we report the establishment of patient derived xenograft (PDX) and cell line models that reflect the genomic and biological heterogeneity of the human disease. Models demonstrate high genomic concordance with the corresponding patient tumors, with invasive tumors more likely to successfully engraft. Treatment of PDX models with chemotherapy recapitulates responses observed in patients. Analysis of a HER2 S310F-mutant PDX suggests that an antibody drug conjugate targeting HER2 would have superior efficacy versus selective HER2 kinase inhibitors. In sum, the biological and phenotypic concordance between patient and PDXs suggest that these models could facilitate studies of intrinsic and acquired resistance and the development of personalized medicine strategies for UTUC patients.

A Phase 1/2 Trial of Ruxolitinib and Erlotinib in Patients with EGFR-Mutant Lung Adenocarcinomas with Acquired Resistance to Erlotinib.

INTRODUCTION: Resistance to EGFR tyrosine kinase inhibitors develops in patients with EGFR-mutant lung cancers. New treatments are needed to address resistance not mediated by EGFR T790M; preclinical evidence suggests that the Janus kinase/signal transducers and activators of transcription signaling pathway is important in acquired resistance to EGFR-directed therapy. METHODS: We evaluated the toxicity and efficacy of erlotinib and ruxolitinib in patients with EGFR-mutant lung cancers with acquired resistance to erlotinib. Exosomes were analyzed to assess changes in relevant protein expression during treatment. RESULTS: We enrolled 22 patients: 12 patients in the phase 1 portion of the study and 10 patients in the phase 2 portion. We did not observe any dose-limiting toxicities. The maximum tolerated dose of erlotinib was 150 mg daily and that of ruxolitinib was 20 mg twice daily. The most frequent toxicities (any grade) were anemia, diarrhea, and elevation of liver function test results. One partial response was observed (5% [95% confidence interval: 0-13]). The median progression-free survival was 2.2 months (95% confidence interval: 1.4-4.1). CONCLUSION: This is the first study assessing the combination of EGFR and Janus kinase inhibition in patients with EGFR-mutant lung cancers. The combination was well tolerated but ineffective. Exosomal EGFR levels may reflect changes in tumor EGFR expression in response to therapy.

JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors.

Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non-small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells' dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC.

Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling.

The signal transducers and activators of transcription 3 (STAT3) signaling pathway plays critical roles in the pathogenesis and progression of various human cancers, including non-small cell lung cancer (NSCLC). In this study, we aimed to evaluate the therapeutic potential of physalin A, a bioactive withanolide derived from Physalis alkekengi var. francheti used in traditional Chinese medicine, was evaluated in human NSCLC cells. Its and determined whether it effect oninhibited both constitutive and induced STAT3 activity, through repressing the phosphorylation levels of JAK2 and JAK3, resulting in anti-proliferation and pro-apoptotic effects on NSCLC cells was also determined, and. theThe antitumor effects of physalin A were also validated usingin an in vivo mouse xenograft models of NSCLC cells. Physalin A had anti-proliferative and pro-apoptotic effects in NSCLC cells with constitutively activated STAT3; it also suppressed both constitutive and induced STAT3 activity by modulating the phosphorylation of JAK2 and JAK3. Furthermore, physalin A abrogated the nuclear translocation and transcriptional activity of STAT3, thereby decreasing the expression levels of STAT3, its target genes, such as Bcl-2 and XIAP. Knockdown of STAT3 expression by small interfering RNA (siRNA) significantly enhanced the pro-apoptotic effects of physalin A in NSCLC cells. Moreover, physalin A significantly suppressed tumor xenograft growth. Thus, as an inhibitor of JAK2/3-STAT3 signaling, physalin A, has potent anti-tumor activities, which may facilitate the development of a therapeutic strategy for treating NSCLC.

Differential interleukin-6/Stat3 signaling as a function of cellular context mediates Ras-induced transformation.

INTRODUCTION: Tyrosine phosphorylated signal transducer and activator of transcription 3 (pStat3) is present in numerous cancers and is required for mediating tumorigenesis. Autocrine and paracrine interleukin (IL)-6 signaling is the principal mechanism by which Stat3 is persistently phosphorylated in epithelial tumors including breast, lung, colon and gastric cancer. The Ras oncogene mediates cellular transformation without evidence of pStat3 in cultured cells. Recently, however non-tyrosine phosphorylated Stat3 was shown to have a transcriptional activating function, a role in mitochondrial function and to mediate cell migration.. Here we examined the role of Stat3 in Ras mediated transformation. METHODS: Ha-rasV12 transformed mammary epithelial cells (MCF10A) cells were transduced with a Stat3shRNA, IL-6shRNA and/or treated with inhibitors of Janus kinases (JAKs) to examine the role of the IL-6 signaling pathway in Ras mediated invasion and tumorigenesis. RESULTS: Cellular migration, invasion, anchorage independent growth and tumorigenesis were largely abrogated in the Stat3-reduced cells compared to control cells. Analysis of MCF10A-Ras tumors revealed high levels of pStat3 and interleukin-6. Tumors derived from transgenic MMTV-K-Ras mice were also found to express pStat3 and IL-6. MCF10A-Ras cells, when grown in a three-dimensional Matrigel culture system revealed the appearance of the junctional protein E-Cadherin as a consequence of reducing Stat3 levels or inhibiting Stat3 activity. Decreasing IL-6 levels in the MCF10A-Ras cells abrogated tumorigenesis and reduced cell migration. By isolating Ras-expressing primary tumors and serially passaging these cells in two-dimensional culture led to a decrease in IL-6 and pStat3 levels with the reappearance of E-Cadherin. CONCLUSIONS: The cellular and environmental context can lead to differential IL-6/pStat3 signaling and a dependency on this cytokine and transcription factor for migration, invasion and tumorigenesis.

Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.

BACKGROUND: The ability of cancer to infiltrate along nerves is a common clinical observation in pancreas, head and neck, prostate, breast, and gastrointestinal carcinomas. For these tumors, nerves may provide a conduit for local cancer progression into the central nervous system. Although neural invasion is associated with poor outcome, the mechanism that triggers it is unknown. METHODS: We used an in vitro Matrigel dorsal root ganglion and pancreatic cancer cell coculture model to assess the dynamic interactions between nerves and cancer cell migration and the role of glial cell-derived neurotrophic factor (GDNF). An in vivo murine sciatic nerve model was used to study how nerve invasion affects sciatic nerve function. RESULTS: Nerves induced a polarized neurotrophic migration of cancer cells (PNMCs) along their axons, which was more efficient than in the absence of nerves (migration distance: mean = 187.1 microm, 95% confidence interval [CI] = 148 to 226 microm vs 14.4 microm, 95% CI = 9.58 to 19.22 microm, difference = 143 microm; P < .001; n = 20). PNMC was induced by secretion of GDNF, via phosphorylation of the RET-Ras-mitogen-activated protein kinase pathway. Nerves from mice deficient in GDNF had reduced ability to attract cancer cells (nerve invasion index: wild type vs gdnf+/-, mean = 0.76, 95% CI = 0.75 to 0.77 vs 0.43, 95% CI = 0.42 to 0.44; P < .001; n = 60-66). Tumor specimens excised from patients with neuroinvasive pancreatic carcinoma had higher expression of the GDNF receptors RET and GRFalpha1 as compared with normal tissue. Finally, systemic therapy with pyrazolopyrimidine-1, a tyrosine kinase inhibitor targeting the RET pathway, suppressed nerve invasion toward the spinal cord and prevented paralysis in mice. CONCLUSION: These data provide evidence for paracrine regulation of pancreatic cancer invasion by nerves, which may have important implications for potential therapy directed against nerve invasion by cancer.