Novel dual action PARP and microtubule polymerization inhibitor AMXI-5001 powerfully inhibits growth of esophageal carcinoma both alone and in combination with radiotherapy.

Esophageal cancer is one of the leading causes of cancer deaths globally with an incidence that is concentrated in specific hot spots in Eastern Asia, the Middle East, Eastern Africa, and South America. 10-year overall survival for patients treated with standard of care chemoradiation followed by surgical resection is below 40% highlighting the need for novel therapeutics to treat this disease. We assessed the effect of AMXI-5001, a novel small molecule poly ADP-Ribose polymerase (PARP) inhibitor and microtubule polymerization inhibitor on tumor growth inhibition in both in-vitro and in-vivo murine models. We found that AMXI-5001 was the most potent growth inhibitor of 8 out of 9 different esophageal carcinoma cell lines compared to other clinically available PARP inhibitors, Olaparib, Niraparib, Rucaparib, and Talazoparib. We then confirmed the previously described mechanism of action of AMXI-5001 as a PARP-inhibitor and microtubule polymerization inhibitor using both a PARP trapping assay and immunofluorescence. To further assess AMXI-5001's potential as a therapeutic for esophageal carcinoma we evaluated the effect of AMXI-5001 in combination with standard chemotherapy agents, Cisplatin and 5 Fluorouracil. We showed that AMXI-5001 synergistically inhibits growth in KYSE-70, a squamous esophageal cell line in combination with these drugs. In addition, we found that AMXI-5001 was an effective radiosensitizer, and squamous esophageal carcinoma cell lines treated 24 hours prior to external beam radiation showed significantly more growth inhibition compared to controls. Finally, we assessed the effect of AMXI-5001 monotherapy and in combination with radiotherapy in a xenograft mouse model implanted with subcutaneous KYSE-70 cells. Compared to vehicle control, and those treated with either AMXI-5001 alone or radiation alone, mice treated with both AMXI-5001 and radiation had significant tumor response. In conclusion, AMXI-5001 is an orally bioavailable dual-action PARP and microtubule polymerization inhibitor that holds promise in the treatment of esophageal carcinoma.

Randomized Phase II Trial Comparing Bevacizumab Plus Carboplatin and Paclitaxel With Carboplatin and Paclitaxel Alone in Previously Untreated Locally Advanced or Metastatic Non-Small-Cell Lung Cancer.

PURPOSE: To investigate the efficacy and safety of bevacizumab plus carboplatin and paclitaxel in patients with advanced or recurrent non-small-cell lung cancer. PATIENTS AND METHODS: In a phase II trial, 99 patients were randomly assigned to bevacizumab 7.5 (n = 32) or 15 mg/kg (n = 35) plus carboplatin (area under the curve = 6) and paclitaxel (200 mg/m2) every 3 weeks or carboplatin and paclitaxel alone (n = 32). Primary efficacy end points were time to disease progression and best confirmed response rate. On disease progression, patients in the control arm had the option to receive single-agent bevacizumab 15 mg/kg every 3 weeks. RESULTS: Compared with the control arm, treatment with carboplatin and paclitaxel plus bevacizumab (15 mg/kg) resulted in a higher response rate (31.5% v 18.8%), longer median time to progression (7.4 v 4.2 months) and a modest increase in survival (17.7 v 14.9 months). Of the 19 control patients that crossed over to single-agent bevacizumab, five experienced stable disease, and 1-year survival was 47%. Bleeding was the most prominent adverse event and was manifested in two distinct clinical patterns; minor mucocutaneous hemorrhage and major hemoptysis. Major hemoptysis was associated with squamous cell histology, tumor necrosis and cavitation, and disease location close to major blood vessels. CONCLUSION: Bevacizumab in combination with carboplatin and paclitaxel improved overall response and time to progression in patients with advanced or recurrent non-small-cell lung cancer. Patients with nonsquamous cell histology appear to be a subpopulation with improved outcome and acceptable safety risks.

Comparative genomics between matched solid and lepidic portions of semi-solid lung adenocarcinomas.

BACKGROUND: Genetic changes that drive the transition from lepidic to invasive cancer development within a radiographic ground glass or semi-solid lung lesion (SSL) are not well understood. Biomarkers to predict the transition to solid, invasive cancer within SSL are needed. METHODS: Patients with surgically resected SSL were identified retrospectively from a surgical database. Clinical characteristics and survival were compared between stage I SSL (n = 65) and solid adenocarcinomas (n = 120) resected during the same time period. Areas of normal lung, in situ lepidic, and invasive solid tumor were microdissected from within the same SSL specimens and next generation sequencing (NGS) and Affymetrix microarray of gene expression were performed. RESULTS: There were more never smokers, Asian patients, and sub-lobar resections among SSL but no difference in 5-year survival between SSL and solid adenocarcinoma. Driver mutations found in both lepidic and solid invasive portion were EGFR (43%), KRAS (21%), and DNMT3A (5%). CEACAM5 was the most upregulated gene found in solid, invasive portions of SSL. Lepidic and invasive solid areas had many similarities in gene expression, however there were some significant differences with the gene SPP1 being a unique biomarker for the invasive component of a SSL. CONCLUSIONS: Common lung cancer driver mutations are present in in situ lepidic as well as invasive solid portions of a SSL, suggesting early development of driver mutations. CEACAM5 and SPP1 emerged as promising biomarkers of invasive potential in semi-solid lesions. Other studies have shown both genes to correlate with poor prognosis in lung cancer and their role in evolution of semi-solid lung lesions warrants further study.

SARS-CoV-2 infection of airway organoids reveals conserved use of Tetraspanin-8 by Ancestral, Delta, and Omicron variants.

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.

MK256 is a novel CDK8 inhibitor with potent antitumor activity in AML through downregulation of the STAT pathway.

Acute myeloid leukemia (AML) is the most lethal form of AML due to disease relapse. Cyclin dependent kinase 8 (CDK8) is a serine/threonine kinase that belongs to the family of Cyclin-dependent kinases and is an emerging target for the treatment of AML. MK256, a potent, selective, and orally available CDK8 inhibitor was developed to target AML. We sought to examine the anticancer effect of MK256 on AML. In CD34+/CD38- leukemia stem cells, we found that MK256 induced differentiation and maturation. Treatment of MK256 inhibited proliferation of AML cell lines. Further studies of the inhibitory effect suggested that MK256 not only downregulated phosphorylated STAT1(S727) and STAT5(S726), but also lowered mRNA expressions of MCL-1 and CCL2 in AML cell lines. Efficacy of MK256 was shown in MOLM-14 xenograft models, and the inhibitory effect on phosphorylated STAT1(S727) and STAT5(S726) with treatment of MK256 was observed in vivo. Pharmacologic dynamics study of MK256 in MOLM-14 xenograft models showed dose-dependent inhibition of the STAT pathway. Both in vitro and in vivo studies suggested that MK256 could effectively downregulate the STAT pathway. In vitro ADME, pharmacological kinetics, and toxicity of MK256 were profiled to evaluate the drug properties of MK256. Our results show that MK256 is a novel CDK8 inhibitor with a desirable efficacy and safety profile and has great potential to be a promising drug candidate for AML through regulating the STAT pathway.

Genetic and immunologic features of recurrent stage I lung adenocarcinoma.

Although surgery for early-stage lung cancer offers the best chance of cure, recurrence still occurs between 30 and 50% of the time. Why patients frequently recur after complete resection of early-stage lung cancer remains unclear. Using a large cohort of stage I lung adenocarcinoma patients, distinct genetic, genomic, epigenetic, and immunologic profiles of recurrent tumors were analyzed using a novel recurrence classifier. To characterize the tumor immune microenvironment of recurrent stage I tumors, unique tumor-infiltrating immune population markers were identified using single cell RNA-seq on a separate cohort of patients undergoing stage I lung adenocarcinoma resection and applied to a large study cohort using digital cytometry. Recurrent stage I lung adenocarcinomas demonstrated higher mutation and lower methylation burden than non-recurrent tumors, as well as widespread activation of known cancer and cell cycle pathways. Simultaneously, recurrent tumors displayed downregulation of immune response pathways including antigen presentation and Th1/Th2 activation. Recurrent tumors were depleted in adaptive immune populations, and depletion of adaptive immune populations and low cytolytic activity were prognostic of stage I recurrence. Genomic instability and impaired adaptive immune responses are key features of stage I lung adenocarcinoma immunosurveillance escape and recurrence after surgery.

The effect of cullin 4A on lung cancer cell chemosensitivity to paclitaxel through p33ING1b regulation.

Cullin 4A (Cul4A) reportedly has oncogenic roles in several cancer types by regulating tumor suppressors through the ubiquitination and proteolysis of the tumor suppressor. In addition, Cul4A is associated with chemosensitivity to chemotherapy drugs. This study investigated the association between Cul4A and lung cancer cell chemosensitivity to paclitaxel, particularly with respect to the role of the p33 inhibitor of the growth 1 (p33ING1b) tumor suppressor. The results showed that the Cul4A knockdown upregulated the p33ING1b expression in lung cancer cells and increased the lung cancer cell and mice tumor xenograft chemosensitivity to paclitaxel. The Cul4A knockdown also inhibited the growth and increased the apoptosis in the tumor xenografts treated with paclitaxel. Notably, the p33ING1b overexpression increased the lung cancer cell chemosensitivity to paclitaxel, but the p33ING1b knockdown reduced the chemosensitivity. A further analysis demonstrated that Cul4A regulates the expression of p33ING1b through protein-protein interactions, ubiquitination, and protein degradation. In conclusion, the present findings suggest that Cul4A mediates the chemosensitivity of lung cancer cells to paclitaxel by regulating p33ING1b. These findings may offer novel insights into future therapeutic strategies for lung cancer that target Cul4A.

Molecular Risk Stratification is Independent of EGFR Mutation Status in Identifying Early-Stage Non-Squamous Non-Small Cell Lung Cancer Patients at Risk for Recurrence and Likely to Benefit From Adjuvant Chemotherapy.

BACKGROUND: A clinically-certified gene expression profile improved survival in a cohort of stage I-IIA NSCLC patients by identifying those likely to benefit from adjuvant intervention. EGFR mutation status has not provided this type of predictive risk discrimination in stage IA NSCLC, and overtreatment of low-risk stage IB patients may have limited the overall benefit seen recently in the adjuvant application of a third-generation TKI. We compared EGFR mutation data to molecular risk stratification in a prospective, early-stage cohort. MATERIALS AND METHODS: Two hundred fifty eligible stage I-IIA non-squamous NSCLC patients underwent prospective molecular risk stratification by the 14-gene prognostic assay. Platinum doublet adjuvant chemotherapy (AC) was recommended for molecular high-risk (MHR). Differences in freedom from recurrence (FFR) and disease-free survival (DFS) were evaluated. RESULTS: At 29 months, prospective molecular testing yielded an estimated FFR of 94.6% and 72.4% in low-risk and untreated MHR patients, respectively, and 97.0% among MHR patients receiving AC (P < .001). In contrast, there was no association between EGFR status and recurrence, while molecular risk predicted survival and response to AC within both the EGFR mutation(+) and mutation(-) populations. Sixty-seven percent of EGFR(+) and 49% of EGFR(-) patients were molecular low-risk. CONCLUSION: This prospective study demonstrates the utility of the 14-gene assay independent of EGFR mutation. Basing adjuvant intervention in early-stage NSCLC on EGFR status alone may undertreat up to 51% of EGFR(-) patients likely to benefit from adjuvant intervention, and overtreat as many as 67% of EGFR(+) patients more likely to be free of residual disease.

Improved outcomes and staging in non-small-cell lung cancer guided by a molecular assay.

There remains a critical need for improved staging of non-small-cell lung cancer, as recurrence and mortality due to undetectable metastases at the time of surgery remain high even after complete resection of tumors currently categorized as 'early stage.' A 14-gene quantitative PCR-based expression profile has been extensively validated to better identify patients at high-risk of 5-year mortality after surgical resection than conventional staging - mortality that almost always results from previously undetectable metastases. Furthermore, prospective studies now suggest a predictive benefit in disease-free survival when the assay is used to guide adjuvant chemotherapy decisions in early-stage non-small-cell lung cancer patients.

Lay abstract There is a need for improvement in the way early-stage non-small-cell lung cancers are staged and treated because many patients with 'early-stage' disease suffer high rates of cancer recurrence after surgery. In recent years, a specialized test has been developed to allow better characterization of a tumor's risk of recurrence based on the genes being expressed by tumor cells. Use of this test, in conjunction with standard staging methods, is better able to identify patients at high risk of cancer recurrence after surgery. Evidence suggests that giving chemotherapy to patients at high risk of recurrence after surgery reduces recurrence rates and improves long-term patient survival.

SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator.

SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2- positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.

Extracellular sulfatases as potential blood-based biomarkers for early detection of lung cancer.

PURPOSE: Non-small lung (NSCLC) is the deadliest cancer, with survival measured in months. Earlier diagnosis using a robust biomarker would likely improve survival. This study aims to determine whether blood levels of the extracellular sulfatases (SULF1 and SULF2) and their bio-activity can serve as novel biomarkers for NSCLC early detection. MATERIALS AND METHODS: Using human plasma specimens from NSCLC patients, nonmalignant COPD patients, and healthy individuals, we determined the association between plasma SULF levels and the presence of NSCLC. We assessed the plasma SULF levels as a function of sex and age. We also evaluated the plasma levels of heparin-binding factors potentially mobilized by the SULFs. To increase test specificity of blood SULF2 as a biomarker for the early diagnosis of NSCLC, we investigated the presence of a tumor-specific SULF2 isoform released in the blood, which could be used as a biomarker alone or in multiplex assays. RESULTS: The median level of plasma SULF2 was significantly elevated in NSCLC patients than in healthy controls (∼2 fold). However, these data were confounded by age. Surprisingly, COPD patients also showed a dramatically increased SULF2 plasma level. We showed a significant increase in the median plasma levels of several HSPG-binding factors in early-stage NSCLC patients compared to controls. Furthermore, we revealed a significant positive correlation of the SULF2 protein level with the plasma levels of two HSPG-binding factors IL6 and IL8. We demonstrated that NSCLC cancer cells and tissues overexpress a SULF2 splice variant. We determined the presence of a SULF2 splice variant form in NSCLC plasma, which was not detectable in COPD and control plasmas. CONCLUSION: Our findings highlight the potential for the plasma levels of SULF2 protein and its bio-activity as novel blood biomarkers for early diagnosis of NSCLC.

Resectability, Recurrence, and Risk Stratification of Giant Solitary Fibrous Tumors in the Thoracic Cavity.

BACKGROUND: Solitary fibrous tumors (SFTs) are rare mesenchymal tumors most commonly arising from the pleura in the thoracic cavity. The impact of tumor size on risk of recurrence in thoracic SFTs is not well understood. METHODS: A single institution review was performed on all resected thoracic SFTs (1992-2019) with giant SFT defined as ≥ 15 cm. Clinical information, pathologic characteristics, and long-term survival data were collected, and predictors of recurrence and survival were evaluated with regression and Kaplan-Meier analysis. RESULTS: There were 38 thoracic SFTs resected from patients, with the majority of tumors (n = 23, 60.5%) originating from visceral pleura. There were nine (23.7%) giant SFTs with a mean size 20.4 cm (range 17-30 cm). Mean follow-up time was 81.0 months (range 1-261 months), during which 4 of 38 (10.5%) patients experienced a recurrence within the thorax (range 51-178 months). The presence of tumor necrosis (p = 0.021) and ≥ 4 mitoses per high-powered field (p = 0.010) were associated with SFT recurrence on univariate regression. Overall 5-year, 10-year, and 20-year survival was 78.2%, 72.6%, and 42.4%, respectively, and SFT-related mortality occurred in three patients at 83, 180, and 208 months postoperatively. There were no recurrences or SFT-related mortality among patients with giant SFT. CONCLUSION: This study represents one of the largest contemporary single institution reviews of long-term outcomes of giant thoracic SFT. Our data suggest that size is not a risk factor for recurrence in thoracic SFTs and long-term survival is excellent for giant SFTs.

Bioinformatic Approaches to Validation and Functional Analysis of 3D Lung Cancer Models.

3D models of cancer have the potential to improve basic, translational, and clinical studies. Patient-derived xenografts, spheroids, and organoids are broad categories of 3D models of cancer, and to date, these 3D models of cancer have been established for a variety of cancer types. In lung cancer, for example, 3D models offer a promising new avenue to gain novel insights into lung tumor biology and improve outcomes for patients afflicted with the number one cancer killer worldwide. However, the adoption and utility of these 3D models of cancer vary, and demonstrating the fidelity of these models is a critical first step before seeking meaningful applications. Here, we review use cases of current 3D lung cancer models and bioinformatic approaches to assessing model fidelity. Bioinformatics approaches play a key role in both validating 3D lung cancer models and high dimensional functional analyses to support downstream applications.

A novel isoform of Homeodomain-interacting protein kinase-2 promotes YAP/TEAD transcriptional activity in NSCLC cells.

Homeodomain-interacting protein kinase-2 (HIPK2) can either promote or inhibit transcription depending on cellular context. In this study, we show that a new HIPK2 isoform increases TEAD reporter activity in NSCLC cells. We detected HIPK2 copy number gain in 5/6 (83.3%) NSCLC cell lines. In NSCLC patients with high HIPK2 mRNA expression in the Human Protein Atlas, the five-year survival rate is significantly lower than in patients with low expression (38% vs 47%; p = 0.047). We also found that 70/78 (89.7%) of NSCLC tissues have moderate to strong expression of the N-terminal HIPK2 protein. We detected and cloned a novel HIPK2 isoform 3 and found that its forced overexpression promotes TEAD reporter activity in NSCLC cells. Expressing HIPK2 isoform 3_K228A kinase-dead plasmid failed to increase TEAD reporter activity in NSCLC cells. Next, we showed that two siRNAs targeting HIPK2 decreased HIPK2 isoform 3 and YAP protein levels in NSCLC cells. Degradation of the YAP protein was accelerated after HIPK2 knockdown in NSCLC cells. Inhibition of HIPK2 isoform 3 decreased the mRNA expression of YAP downstream gene CTGF. The specific HIPK2 kinase inhibitor TBID decreased TEAD reporter activity, reduced cancer side populations, and inhibited tumorsphere formation of NSCLC cells. In summary, this study indicates that HIPK2 isoform 3, the main HIPK2 isoform expressed in NSCLC, promotes YAP/TEAD transcriptional activity in NSCLC cells. Our results suggest that HIPK2 isoform 3 may be a potential therapeutic target for NSCLC.

Perioperative Lung Resection Outcomes After Implementation of a Multidisciplinary, Evidence-based Thoracic ERAS Program.

OBJECTIVE: This prospective study evaluated perioperative lung resection outcomes after implementation of a multidisciplinary, evidence-based Thoracic Enhanced Recovery After Surgery (ERAS) Program in an academic, quaternary-care center. BACKGROUND: ERAS programs have the potential to improve outcomes, but have not been widely utilized in thoracic surgery. METHODS: In all, 295 patients underwent elective lung resection for pulmonary malignancy from 2015 to 2019 PRE (n = 169) and POST (n = 126) implementation of an ERAS program containing all major ERAS Society guidelines. Propensity score-matched analysis, based upon patient, tumor, and surgical characteristics, was utilized to evaluate outcomes. RESULTS: After ERAS implementation, there was increased minimally invasive surgery (PRE 39.6%→POST 62.7%), reduced intensive care unit utilization (PRE 70.4%→POST 21.4%), improved chest tube (PRE 24.3%→POST 54.8%) and urinary catheter (PRE 20.1%→POST 65.1%) removal by postoperative day 1, and increased ambulation ≥3× on postoperative day 1 (PRE 46.8%→POST 54.8%). Propensity score-matched analysis that accounted for minimally invasive surgery demonstrated that program implementation reduced length of stay by 1.2 days [95% confidence interval (CI) 0.3-2.0; PRE 4.4→POST 3.2), morbidity by 12.0% (95% CI 1.6%-22.5%; PRE 32.0%→POST 20.0%), opioid use by 19 oral morphine equivalents daily (95% CI 1-36; PRE 101→POST 82), and the direct costs of surgery and hospitalization by $3500 (95% CI $1100-5900; PRE $23,000→POST $19,500). Despite expedited discharge, readmission remained unchanged (PRE 6.3%→POST 6.6%; P = 0.94). CONCLUSIONS: The Thoracic ERAS Program for lung resection reduced length of stay, morbidity, opioid use, and direct costs without change in readmission. This is the first external validation of the ERAS Society thoracic guidelines; adoption by other centers may show similar benefit.

Ponatinib is a potential therapeutic approach for malignant pleural mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is a rare and deadly malignancy. Current MPM therapies remain inadequate, and outcomes are often disappointing. New meaningful therapeutic approaches are urgently needed. Accumulating evidence indicates that the cAbl pathway promotes various tumor-stimulating processes in MPM. In this study, we sought to determine ponatinib's potential utility, a clinically approved and potent cAbl inhibitor, in MPM treatment. MATERIAL AND METHODS: Four MPM lines (MSTO211H, H28, H2452, H2052) were treated with ponatinib in vitro, and their growth was assessed. Scratch wound assay was used to investigate the ponatinib effect on cell migration. The expression levels of pAbl and its downstream effectors pCrkL, pAKT, and pSTAT5 were characterized. The in vivo ponatinib effect was evaluated in human MPM cells derived tumor model. RESULTS: In all four MPM lines, significant expression levels of phosphorylated cAbl/Arg and pCrkl were observed. Differentially but strongly, ponatinib inhibited the in vitro cell growth and migration of all four MPM line. Western blot analysis showed that the activation of Abl signaling was blocked in the ponatinib-treated MMP lines. In keeping, the cellular levels of pAbl and its downstream effector pCrkL, pAKT, and pSTAT5 were markedly decrease following ponatinib treatment. Moreover, ponatinib treatment amplified the levels of γH2AX in cells denoting increased double-strand DNA breaks levels. Notably, ponatinib treatment reduced in vivo tumor growth and reduced pCrkl and pSTAT5 levels in tumor samples. CONCLUSION: Ponatinib may offer a new therapeutic strategy for MPM patients based on cAbl signaling pathway inhibition.

Development of novel monoclonal antibodies and immunoassays for sensitive and specific detection of SULF1 endosulfatase.

BACKGROUND: Cell-surface heparan sulfate proteoglycans (HSPGs) function as receptors or co-receptors for ligand binding and mediate the transmission of critical extracellular signals into cells. The complex and dynamic modifications of heparan sulfates on the core proteins are highly regulated to achieve precise signaling transduction. Extracellular endosulfatase Sulf1 catalyzes the removal of 6-O sulfation from HSPGs and thus regulates signaling mediated by 6-O sulfation on HSPGs. The expression of Sulf1 is altered in many cancers. Further studies are needed to clarify Sulf1 role in tumorigenesis, and new tools that can expand our knowledge in this field are required. METHODS: We have developed and validated novel SULF1 monoclonal antibodies (mAbs). The isotype and subclass for each of these antibodies were determined. These antibodies provide invaluable reagents to assess SULF1- tissue and blood levels by immunohistochemistry and ELISA assays, respectively. RESULTS: This study reports novel mAbs and immunoassays developed for sensitive and specific human Sulf1 protein detection. Using these SULF1 mAbs, we developed an ELISA assay to investigate whether blood-derived SULF1 may be a useful biomarker for detecting cancer early. Furthermore, we have demonstrated the utility of these antibodies for Sulf1 protein detection, localization, and quantification in biospecimens using various immunoassays. CONCLUSIONS: This study describes novel Sulf1 mAbs suitable for various immunoassays, including Western blot analysis, ELISA, and immunohistochemistry, which can help understand Sulf1 pathophysiological role. GENERAL SIGNIFICANCE: New tools to assess and clarify SULF1 role in tumorigenesis are needed. Our novel Sulf1 mAbs and immunoassays assay may have utility for such application.

Evaluation of a National Comprehensive Cancer Network Guidelines-Based Decision Support Tool in Patients With Non-Small Cell Lung Cancer: A Nonrandomized Clinical Trial.

Importance: The association of guideline-based decision support with the quality of care in patients with non-small cell lung cancer (NSCLC) is not known. Objective: To evaluate the association of exposure to the National Comprehensive Cancer Center (NCCN) guidelines with guideline-concordant care and patients' decisional conflict. Design, Setting, and Participants: A nonrandomized clinical trial, conducted at a tertiary care academic institution, enrolled patients from February 23, 2015, to September 28, 2017. Data analysis was conducted from July 19, 2019, to April 22, 2020. A cohort of 76 patients with NSCLC seen at diagnosis or disease progression and a retrospective cohort of 157 patients treated before the trial were included. Adherence to 6 NCCN recommendations were evaluated: (1) smoking cessation counseling, (2) adjuvant chemotherapy for patients with stage IB to IIB NSCLC after surgery, (3) pathologic mediastinal staging in patients with stage III NSCLC before surgery, (4) pathologic mediastinal staging in patients with stage III NSCLC before nonsurgical treatment, (5) definitive chemoradiotherapy for patients with stage III NSCLC not having surgery, and (6) molecular testing for epidermal growth factor receptor and anaplastic lymphoma kinase alterations for patients with stage IV NSCLC. Subgroup analysis was conducted to compare the rates of guideline concordance between the prospective and retrospective cohorts. Secondary end points included decisional conflict and satisfaction. Interventions: An online tool customizing the NCCN guidelines to patients' clinical and pathologic features was used during consultation, facilitated by a trained coordinator. Main Outcomes and Measures: Concordance of practice with 6 NCCN treatment recommendations on NSCLC and patients' decisional conflict. Results: Of the 76 patients with NSCLC, 44 were men (57.9%), median age at diagnosis was 68 years (interquartile range [IQR], 41-87 years), and 59 patients (77.6%) had adenocarcinoma. In the retrospective cohort, 91 of 157 patients (58.0%) were men, median age at diagnosis was 66 years (IQR, 61-65 years), and 105 patients (66.9%) had adenocarcinoma. After the intervention, patients received more smoking cessation counseling (4 of 5 [80.0%] vs 1 of 24 [4.2%], P < .001) and less adjuvant chemotherapy (0 of 7 vs 7 of 11 [63.6%]; P = .012). There was no significant change in mutation testing of non-squamous cell stage IV disease (20 of 20 [100%] vs 48 of 57 [84.2%]; P = .10). There was no significant change in pathologic mediastinal staging or initial chemoradiotherapy for patients with stage III disease. After consultation with the tool, decisional conflict scores improved by a median of 20 points (IQR, 3-34; P < .001). Conclusions and Relevance: The findings of this study suggest that exposure to the NCCN guidelines is associated with increased guideline-concordant care for 2 of 6 preselected recommendations and improvement in decisional conflict. Trial Registration: ClinicalTrials.gov Identifier: NCT03982459.

AMXI-5001, a novel dual parp1/2 and microtubule polymerization inhibitor for the treatment of human cancers.

Poly (ADP-ribose) polymerase (PARP) has recently emerged as a central mediator in cancer resistance against numerous anticancer agents to include chemotherapeutic agents such as microtubule targeting agents and DNA damaging agents. Here, we describe AMXI-5001, a novel, highly potent dual PARP1/2 and microtubule polymerization inhibitor with favorable metabolic stability, oral bioavailability, and pharmacokinetic properties. The potency and selectivity of AMXI-5001 were determined by biochemical assays. Anticancer activity either as a single-agent or in combination with other antitumor agents was evaluated in vitro. In vivo antitumor activity as a single-agent was assessed in a triple-negative breast cancer (TNBC) model. AMXI-5001 demonstrates comparable IC50 inhibition against PARP and microtubule polymerization as clinical PARP inhibitors (Olaparib, Rucaparib, Niraparib, and Talazoparib) and the potent polymerization inhibitor (Vinblastine), respectively. In vitro, AMXI-5001 exhibited selective antitumor cytotoxicity across a wide variety of human cancer cells with much lower IC50s than existing clinical PARP1/2 inhibitors. AMXI-5001 is highly active in both BRCA mutated and wild type cancers. AMXI-5001 is orally bioavailable. AMXI-5001 elicited a remarkable In vivo preclinical anti-tumor activity in a BRCA mutated TNBC model. Oral administration of AMXI-5001 induced complete regression of established tumors, including exceedingly large tumors. AMXI-5001 resulted in superior anti-tumor effects compared to either single agent (PARP or microtubule) inhibitor or combination with both agents. AMXI-5001 will enter clinical trial testing soon and represents a promising, novel first in class dual PARP1/2 and microtubule polymerization inhibitor that delivers continuous and synchronous one-two punch cancer therapy with one molecule.

The Crosstalk between Src and Hippo/YAP Signaling Pathways in Non-Small Cell Lung Cancer (NSCLC).

The advancement of new therapies, including targeted therapies and immunotherapies, has improved the survival of non-small-cell lung cancer (NSCLC) patients in the last decade. Some NSCLC patients still do not benefit from therapies or encounter progressive disease during the course of treatment because they have intrinsic resistance, acquired resistance, or lack a targetable driver mutation. More investigations on the molecular biology of NSCLC are needed to find useful biomarkers for current therapies and to develop novel therapeutic strategies. Src is a non-receptor tyrosine kinase protein that interacts with cell surface growth factor receptors and the intracellular signaling pathway to maintain cell survival tumorigenesis in NSCLC. The Yes-associated protein (YAP) is one of the main effectors of the Hippo pathway and has been identified as a promoter of drug resistance, cancer progression, and metastasis in NSCLC. Here, we review studies that have investigated the activation of YAP as mediated by Src kinases and demonstrate that Src regulates YAP through three main mechanisms: (1) direct phosphorylation; (2) the activation of pathways repressing Hippo kinases; and (3) Hippo-independent mechanisms. Further work should focus on the efficacy of Src inhibitors in inhibiting YAP activity in NSCLC. In addition, future efforts toward developing potentially reasonable combinations of therapy targeting the Src-YAP axis using other therapies, including targeted therapies and/or immunotherapies, are warranted.