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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

Supplementary Methods

This supplementary data file contains 5 figures pertaining to the tumor distribution of MEK1 hotspot mutations, functional characterization of further MEK1/2 mutants with and without MEK or ERK inhibitor treatment, and the sequence paralogy alignment of MEK1 and MEK2. This file also contains 4 tables detailing hotspot analysis q values for MEK1/2 mutants, tumor incidence of MEK1 in-frame deletions, and a summary of MEK1/2 paralogous residues and their concordant activation status.

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

Bladder cancer (BLCA) is the sixth most common cancer in the U.S with 83,730 new cases, accounting for 17,200 deaths in the year 2021 and 212,536 deaths worldwide. EP300, a histone acetyltransferase gene, is mutated in ~17% of BLCA and in other cancer types such as endometrial cancer. The mechanisms by which EP300 mutations contribute to tumorigenicity of BLCA is not currently understood. To determine the phenotypic effects of loss-of-function EP300 mutations, we generated isogenic BLCA cells with EP300 knocked out (KO) using CRISPR/CAS9. Conspicuous changes in phenotypes consistent with more aggressive forms of cancer were observed in EP300-null BLCA clones, including luminal-basal histotype plasticity, anchorage-independent growth, enhanced in vitro and in vivo cell proliferation and enhanced invasion in Boyden chamber assays. To identify mechanisms whereby EP300 KO promotes increased oncogenicity, we performed phospho Tandem Tag Mass Spectrometry (TMT MS) using lysates from parental and EP300 KO cells. A notable finding was significantly increased expression of phosphorylated STAT3 (pSTAT3), which we subsequently confirmed by western blot. siRNA knockdown and selective inhibitor experiments indicated JAK1 as the upstream activator of pSTAT3 in BLCA cells. Studies of conditioned media (CM) from the EP300 KO cells using ELISA and neutralizing antibodies demonstrated that secreted IL-6 ligand and soluble IL-6R in the CM drove STAT3 activation through the IL-6 family common signaling subunit, gp130, i.e. trans-signaling. BLCA RT112 cells express an oncogenic FGFR3-TACC3 fusion and are sensitive to the FDA-approved FGFR kinase inhibitor erdafitinib. pSTAT3 expression was not dependent on FGFR3 signaling in EP300 KO RT112 cells, and EP300 KO was sufficient to confer erdafitinib resistance in a BLCA context. In sum, our results uncover that EP300 loss enhances IL-6/JAK/STAT3 signaling which promotes BLCA tumorigenesis and FGFR inhibitor resistance. Our findings elucidate a role for cytokine induced sterile inflammation in EP300-mediated tumorigenesis in bladder cancers and suggest that targeting the IL-6/JAK/STAT3 axis may represent a novel therapy strategy to overcome FGFR3 inhibitor resistance. Citation Format: Sizhi P. Gao, James A. Rodrigues, Amanda R. Sabel, Jiaqian Luo, Chen Ziyu, Xinran Tang, Eduardo A. Mascareno, Naryan Rustgi, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene J. Pietzak, Gopakumar V. Iyer, David B. Solit. EP300 loss drives tumorigenesis in bladder cancer via activation of IL-6/JAK/STAT3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 330.

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

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

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., The advancement of upper tract urothelial carcinoma (UTUC) research is hampered by the lack of disease-specific models. Here, the authors report patient derived xenograft and cell line models of UTUC, and show that these models retain the genomic and biological heterogeneity of human disease.

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.

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.

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.

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.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.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.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.

One of the greatest challenges in studying the genomic basis of human cancer is distinguishing the few mutations that directly contribute to tumorigenesis (“drivers”) from the many biologically neutral mutations (“passengers”). Existing methods can identify highly recurrent individual mutations or mutated genes, but capturing functional yet infrequent “long-tail” mutations remains challenging. While individually uncommon, these long-tail mutations are present in as many as one-third of cancer patients, and for many of them targeted therapies currently exist. Therefore, improved approaches to identify individual driver mutations in the long tail will facilitate our understanding of their potentially diverse biological and clinical significance. Some of these mutations can be identified as hotspots based on their recurrence on or around the same residue. Others may occur in different regions but are actually close in protein three-dimensional structures. We have developed a novel method that identifies mutations that significantly cluster together within three-dimensional proximity in protein structures, or 3D hotspots. We applied this method to a combined collection of mutation data of 21,000 sequenced tumor samples in 74 cancers. Our analysis confirmed many well-studied 3D hotspots of functional mutations in cancer genes such as KRAS, BRAF, IDH2, SMAD4, FBXW7, SPOP, RHOA, and PTPN11. Most of these genes have well known individual hotspots, but our analysis suggests that additional mutations in other residues of the protein are adjacent in the folded protein structure and may also be oncogenic. We also identified novel 3D hotspots in known cancer genes such as EP300, MAP2K1, and KDR, as well as several other genes of unknown significance that harbor 3D hotspots (e.g., DCC). Most of these hotspots are present in multiple cancers. To explore the functional consequences and potential translational significance of long-tail low-incidence mutations identified by our method, we assessed several MAP2K1 mutations in vitro. MAP2K1 (MEK1) is a critical effector of MAPK signaling, harbors conventional single-codon hotspots in several cancer types, and a number of selective MAPK pathway inhibitors are either FDA-approved or are being investigated in early-phase clinical trials. Our experiments confirmed that these mutations activate MAP2K1 and confer sensitivity to MAP2K1 inhibition. We have provided an implementation of the algorithm via an interactive web resource connecting to cBioPortal for Cancer Genomics (http://cbioportal.org/) for easy interpretation of cancer genomics data in the context of three-dimensional structures. By adding to cBioPortal's already powerful capacity of clinical decision support, our method and analysis provide a useful approach of interpretation, prioritization and extension of biologically significant and clinically actionable mutations in cancer. Citation Format: Jianjiong Gao, Matthew T. Chang, Brooke E. Sylvester, Hannah C. Johnsen, Sizhi P. Gao, S. Onur Sumer, David B. Solit, Barry S. Taylor, Nikolaus Schultz, Chris Sander. Identification of oncogenic mutation hotspots via three-dimensional proximity. [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 3606.

Non-small cell lung cancer (NSCLC) is the leading cause of death in the United States. A fraction of these tumors harbor somatic activating mutations in the epidermal growth factor receptor (EGFR) leading to constitutive activation of the kinase. Targeting this pathway using a class of tyrosine kinase inhibitors (TKI) such as gefitinib or erlotinib induces significant initial responses in patients with NSCLC expressing activating EGFR mutations, however, disease progression invariably occurs in part due to the outgrowth of cancers with EGFR TKI resistant secondary mutations. There are many ongoing attempts to overcome this problem, but the current efforts are mostly focused on EGFR itself and their effectiveness remains inconclusive. The interleukin-6 (IL-6) /Jak/Stat3 signaling pathway is critical not only for inflammation and immunity, but also for oncogenesis. Stat3 is persistently activated in many human cancers, promoting tumorigenesis through the regulation of genes important for proliferation, apoptosis, invasion, angiogenesis and suppression of anti-tumor immunity. We previously determined that Stat3 is activated in mutant EGFR expressing NSCLC cell lines and primary tumors in part through autocrine upregulation of the IL-6 gene that activates the gp130/Jak signaling. In our current report, we studied the in vivo effect of inhibiting the IL-6/Jak/Stat3 signaling in EGFR TKI-resistant models of NSCLC. Our data demonstrated that Jak inhibition by a novel orally available Jak inhibitor markedly repressed in vivo growth of 3 different TKI resistant NSCLC cell lines as well as a TKI sensitive cell line. Immunohistochemical analyses of these samples revealed reduced proliferation and angiogenesis in Jak inhibitor treated tumors. In addition, administration of an IL-6R blocking antibody to xenograft tumors of a TKI resistant NSCLC cell line also achieved significant reduction in tumor growth compared to controls. We further tested the potential role of IL-6 in a transgenic murine model of TKI-resistant, mutant EGFR mediated lung cancer. Our results demonstrated mice lacking IL-6 still developed lung tumors but at a much slower rate of tumor progression compared to IL-6 expressing littermates. Additional examinations showed that mice lacking IL-6 developed tumors with papillary features compared to IL-6 expressing littermates whereas most of the tumors were of the more advanced, solid/pleiomorphic subtype. IL-6 deficient lung tumors had decreased proliferation and angiogenesis compared to tumors from wildtype littlermates. In summary, our findings lead us to hypothesize that the IL-6/Jak pathway should be considered as a novel therapeutic target for this disease, especially TKI resistant NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4689. doi:10.1158/1538-7445.AM2011-4689