PROTAC-Mediated Dual Degradation of BCL-xL and BCL-2 Is a Highly Effective Therapeutic Strategy in Small-Cell Lung Cancer.

BCL-xL and BCL-2 are validated therapeutic targets in small-cell lung cancer (SCLC). Targeting these proteins with navitoclax (formerly ABT263, a dual BCL-xL/2 inhibitor) induces dose-limiting thrombocytopenia through on-target BCL-xL inhibition in platelets. Therefore, platelet toxicity poses a barrier in advancing the clinical translation of navitoclax. We have developed a strategy to selectively target BCL-xL in tumors, while sparing platelets, by utilizing proteolysis-targeting chimeras (PROTACs) that hijack the cellular ubiquitin proteasome system for target ubiquitination and subsequent degradation. In our previous study, the first-in-class BCL-xL PROTAC, called DT2216, was shown to have synergistic antitumor activities when combined with venetoclax (formerly ABT199, BCL-2-selective inhibitor) in a BCL-xL/2 co-dependent SCLC cell line, NCI-H146 (hereafter referred to as H146), in vitro and in a xenograft model. Guided by these findings, we evaluated our newly developed BCL-xL/2 dual degrader, called 753b, in three BCL-xL/2 co-dependent SCLC cell lines and the H146 xenograft models. 753b was found to degrade both BCL-xL and BCL-2 in these cell lines. Importantly, it was considerably more potent than DT2216, navitoclax, or DT2216 + venetoclax in reducing the viability of BCL-xL/2 co-dependent SCLC cell lines in cell culture. In vivo, 5 mg/kg weekly dosing of 753b was found to lead to significant tumor growth delay, similar to the DT2216 + venetoclax combination in H146 xenografts, by degrading both BCL-xL and BCL-2. Additionally, 753b administration at 5 mg/kg every four days induced tumor regressions. At this dosage, 753b was well tolerated in mice, without observable induction of severe thrombocytopenia as seen with navitoclax, and no evidence of significant changes in mouse body weights. These results suggest that the BCL-xL/2 dual degrader could be an effective and safe therapeutic for a subset of SCLC patients, warranting clinical trials in future.

PROTAC-mediated dual degradation of BCL-xL and BCL-2 is a highly effective therapeutic strategy in small-cell lung cancer.

BCL-xL and BCL-2 are validated therapeutic targets in small-cell lung cancer (SCLC). Targeting these proteins with navitoclax (formerly ABT263, a dual BCL-xL/2 inhibitor) induces dose-limiting thrombocytopenia through on-target BCL-xL inhibition in platelets. Therefore, platelet toxicity poses a barrier in advancing the clinical translation of navitoclax. We have developed a strategy to selectively target BCL-xL in tumors, while sparing platelets, by utilizing proteolysis-targeting chimeras (PROTACs) that hijack the cellular ubiquitin proteasome system for target ubiquitination and subsequent degradation. In our previous study, the first-in-class BCL-xL PROTAC, called DT2216, was shown to have synergistic antitumor activities when combined with venetoclax (formerly ABT199, BCL-2-selective inhibitor) in a BCL-xL/2 co-dependent SCLC cell line, NCI-H146 (hereafter referred to as H146), in vitro and in a xenograft model. Guided by these findings, we evaluated our newly developed BCL-xL/2 dual degrader, called 753b, in three BCL-xL/2 co-dependent SCLC cell lines and the H146 xenograft models. 753b was found to degrade both BCL-xL and BCL-2 in these cell lines. Importantly, it was considerably more potent than DT2216, navitoclax, or DT2216+venetoclax to reduce the viability of BCL-xL/2 co-dependent SCLC cell lines in cell culture. In vivo, 5 mg/kg weekly dosing of 753b leads to significant tumor growth delay similar to the DT2216+venetoclax combination in H146 xenografts by degrading both BCL-xL and BCL-2. Additionally, 753b administration at 5 mg/kg every four days induced tumor regressions. 753b at this dosage was well tolerated in mice without induction of severe thrombocytopenia as seen with navitoclax nor induced significant changes in mouse body weights. These results suggest that the BCL-xL/2 dual degrader could be an effective and safe therapeutic for a subset of SCLC patients warranting clinical trials in future.

Nivolumab monotherapy or combination with ipilimumab with or without cobimetinib in previously treated patients with pancreatic adenocarcinoma (CheckMate 032).

BACKGROUND: Pancreatic cancer is one of the deadliest cancer types and represents a major unmet medical need. CheckMate 032 investigated safety and efficacy of nivolumab monotherapy and nivolumab plus ipilimumab with/without cobimetinib in advanced/metastatic solid tumors, including pancreatic cancer. METHODS: In the original pancreatic cancer cohort, previously treated patients (≥1 prior regimen) with advanced/metastatic pancreatic adenocarcinoma were assigned to nivolumab 3 mg/kg every 2 weeks (monotherapy arm) or nivolumab 1 mg/kg and ipilimumab 1 mg/kg or 3 mg/kg every 3 weeks for four doses, followed by nivolumab 3 mg/kg every 2 weeks (combination arm). A subsequent modified pancreatic cohort (one or two prior regimens) received nivolumab 3 mg/kg every 2 weeks, ipilimumab 1 mg/kg every 6 weeks, and cobimetinib 60 mg orally once daily for 21 days on and 7 days off (triplet arm). The primary endpoint was investigator-assessed objective response rate (ORR). Secondary endpoints were investigator-assessed progression-free survival (PFS), PFS rate, overall survival (OS), OS rate, safety, and tolerability. Additionally, ORR, PFS, and duration of response were assessed by blinded independent central review (BICR) in the triplet arm. RESULTS: 18 patients received nivolumab monotherapy, 21 received nivolumab plus ipilimumab, and 30 received nivolumab plus ipilimumab plus cobimetinib. In the triplet arm, partial responses were observed in two patients per investigator (ORR 6.7% (95% CI 0.8% to 22.1%)) and in three patients per BICR (ORR 10% (95% CI 2.1% to 26.5%)); no responses were observed in the other arms. Median (95% CI) PFS per investigator was 1.4 (1.3 to 2.0), 1.4 (1.2 to 2.7), and 3.0 (1.5 to 4.1) months for the monotherapy, nivolumab plus ipilimumab, and triplet arms, respectively. Median (95% CI) OS was 5.1 (2.0 to 9.0) months, 4.0 (1.9 to 5.6) months, and 6.2 (3.9 to 11.4) months, respectively. Most treatment-related adverse events were grade 2 or less. CONCLUSIONS: Nivolumab with or without ipilimumab did not elicit objective responses in previously treated patients with advanced pancreatic adenocarcinoma, although three confirmed partial responses and manageable safety were observed with cobimetinib-containing triplet therapy. The small sample size and differences in baseline disease-specific characteristics between arms limit interpretation of these results.

Disparities Contributing to Late-Stage Diagnosis of Lung, Colorectal, Breast, and Cervical Cancers: Rural and Urban Poverty in Florida.

Despite advances in cancer screening, late-stage cancer diagnosis is still a major cause of morbidity and mortality in the United States. In this study, we aim to understand demographic and geographic factors associated with receiving a late-stage diagnosis (LSD) of lung, colorectal, breast, or cervical cancer. (1) Methods: We analyzed data of patients with a cancer diagnosis between 2016 and 2020 from the Florida Cancer Data System (FCDS), a statewide population-based registry. To investigate correlates of LSD, we estimated multi-variable logistic regression models for each cancer while controlling for age, sex, race, insurance, and census tract rurality and poverty. (2) Results: Patients from high-poverty rural areas had higher odds for LSD of lung (OR = 1.23, 95% CI (1.10, 1.37)) and breast cancer (OR = 1.31, 95% CI (1.17,1.47)) than patients from low-poverty urban areas. Patients in high-poverty urban areas saw higher odds of LSD for lung (OR = 1.05 95% CI (1.00, 1.09)), breast (OR = 1.10, 95% CI (1.06, 1.14)), and cervical cancer (OR = 1.19, 95% CI (1.03, 1.37)). (3) Conclusions: Financial barriers contributing to decreased access to care likely drive LSD for cancer in rural and urban communities of Florida.

TYMS promotes genomic instability and tumor progression in Ink4a/Arf null background.

We previously showed that elevated TYMS exhibits oncogenic properties and promotes tumorigenesis after a long latency, suggesting cooperation with sequential somatic mutations. Here we report the cooperation of ectopic expression of human TYMS with loss of Ink4a/Arf, one of the most commonly mutated somatic events in human cancer. Using an hTS/Ink4a/Arf -/- genetically engineered mouse model we showed that deregulated TYMS expression in Ink4a/Arf null background accelerates tumorigenesis and metastasis. In addition, tumors from TYMS-expressing mice were associated with a phenotype of genomic instability including enhanced double strand DNA damage, aneuploidy and loss of G1/S checkpoint. Downregulation of TYMS in vitro decreased cell proliferation and sensitized tumor cells to antimetabolite chemotherapy. In addition, depletion of TYMS in vivo by TYMS shRNA reduced tumor incidence, delayed tumor progression and prolonged survival in hTS/Ink4a/Arf -/- mice. Our data shows that activation of TYMS in Ink4a/Arf null background enhances uncontrolled cell proliferation and tumor growth, supporting the development of new agents and strategies targeting TYMS to delay tumorigenesis and prolong survival.

First-in-class multifunctional TYMS nonclassical antifolate inhibitor with potent in vivo activity that prolongs survival.

Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.

Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice.

Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-XL and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on-target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-XL degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co-dependent on BCL-XL and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-XL/MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-XL degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-XL and MCL-1.

PROTAC therapy as a new targeted therapy for lung cancer.

Despite recent advances in molecular therapeutics, lung cancer is still a leading cause of cancer deaths. Currently, limited targeted therapy options and acquired drug resistance present significant barriers in the treatment of patients with lung cancer. New strategies in drug development, including those that take advantage of the intracellular ubiquitin-proteasome system to induce targeted protein degradation, have the potential to advance the field of personalized medicine for patients with lung cancer. Specifically, small molecule proteolysis targeting chimeras (PROTACs), consisting of two ligands connected by a linker that bind to a target protein and an E3 ubiquitin ligase, have been developed against many cancer targets, providing promising opportunities for advanced lung cancer. In this review, we focus on the rationale for PROTAC therapy as a new targeted therapy and the current status of PROTAC development in lung cancer.

Thymidylate synthase accelerates Men1-mediated pancreatic tumor progression and reduces survival.

Clinical studies of cancer patients have shown that overexpression or amplification of thymidylate synthase (TS) correlates with a worse clinical outcome. We previously showed that elevated TS exhibits properties of an oncogene and promotes pancreatic neuroendocrine tumors (PanNETs) with a long latency. To study the causal impact of elevated TS levels in PanNETs, we generated a mouse model with elevated human TS (hTS) and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1-/-). We demonstrated that increased hTS expression was associated with earlier tumor onset and accelerated PanNET development in comparison with control Men1-/- and Men1+/ΔN3-8 mice. We also observed a decrease in overall survival of hTS/Men1+/- and hTS/Men1-/- mice as compared with control mice. We showed that elevated hTS in Men1-deleted tumor cells enhanced cell proliferation, deregulated cell cycle kinetics, and was associated with a higher frequency of somatic mutations, DNA damage, and genomic instability. In addition, we analyzed the survival of 88 patients with PanNETs and observed that high TS protein expression independently predicted worse clinical outcomes. In summary, elevated hTS directly participates in promoting PanNET tumorigenesis with reduced survival in Men1-mutant background. This work will refocus attention on new strategies to inhibit TS activity for PanNET treatment.

Personalised Lung Cancer Screening (PLuS) study to assess the importance of coexisting chronic conditions to clinical practice and policy: protocol for a multicentre observational study.

INTRODUCTION: Lung cancer is the leading cause of cancer death in the USA and worldwide, and lung cancer screening (LCS) with low-dose CT (LDCT) has the potential to improve lung cancer outcomes. A critical question is whether the ratio of potential benefits to harms found in prior LCS trials applies to an older and potentially sicker population. The Personalised Lung Cancer Screening (PLuS) study will help close this knowledge gap by leveraging real-world data to fully characterise LCS recipients. The principal goal of the PLuS study is to characterise the comorbidity burden of individuals undergoing LCS and quantify the benefits and harms of LCS to enable informed decision-making. METHODS AND ANALYSIS: PLuS is a multicentre observational study designed to assemble an LCS cohort from the electronic health records of ~40 000 individuals undergoing annual LCS with LDCT from 2016 to 2022. Data will be integrated into a unified repository to (1) examine the burden of multimorbidity by race/ethnicity, socioeconomic status and age; (2) quantify potential benefits and harms; and (3) use the observational data with validated simulation models in the Cancer Intervention and Surveillance Modeling Network (CISNET) to provide LCS outcomes in the real-world US population. We will fit a multivariable logistic regression model to estimate the adjusted ORs of comorbidity, functional limitations and impaired pulmonary function adjusted for relevant covariates. We will also estimate the cumulative risk of LCS outcomes using discrete-time survival models. To our knowledge, this is the first study to combine observational data and simulation models to estimate the long-term impact of LCS with LDCT. ETHICS AND DISSEMINATION: The study was approved by the Kaiser Permanente Southern California Institutional Review Board and VA Portland Health Care System. The results will be disseminated through publications and presentations at national and international conferences. Safety considerations include protection of patient confidentiality.

Reducing Chemotherapy-Induced DNA Damage via nAChR-Mediated Redox Reprograming-A New Mechanism for SCLC Chemoresistance Boosted by Nicotine.

Up to 60% of patients with small cell lung cancer (SCLC) continue to smoke, which is associated with worse clinical outcomes. Platinum-based chemotherapies, in combination with topoisomerase inhibitors, are first-line therapies for SCLC, with rapid chemoresistance as a major barrier. We provided evidence in this study that nicotine and its major metabolite, cotinine, at physiologically relevant concentrations, reduced the efficacy of platinum-based chemotherapies and facilitated chemoresistance in SCLC cells. Mechanistically, nicotine or cotinine reduced chemotherapy-induced DNA damage by modulating cellular redox processes, with nAChRs as the upstream targets. Surprisingly, cisplatin treatment alone also increased the levels of nAChRs in SCLC cells, which served as a self-defense mechanism against platinum-based therapies. These discoveries were confirmed in long-term in vitro and in vivo studies. Collectively, our results depicted a novel and clinically important mechanism of chemoresistance in SCLC treatment: nicotine exposure significantly compromises the efficacy of platinum-based chemotherapies in SCLC treatment by reducing therapy-induced DNA damage and accelerating chemoresistance acquisition. The results also emphasized the urgent need for tobacco cessation and the control of NRT use for SCLC management.

Managing stage 4 ameloblastoma with dual BRAF/MEK inhibition: A case report with 8-year clinical follow-up.

We present 8-year follow-up on the first patient with stage 4 ameloblastoma carrying a BRAF V600E mutation treated with dual BRAF/MEK inhibition (BRAF/MEKi). He experienced a durable clinical response while on dabrafenib (BRAFi) and trametinib (MEKi) without toxicity nor evidence for drug-resistant tumor progression. He was asymptomatic when he self-discontinued therapy after 4 years of sustained clinical response. He did not return for follow-up until 2.5 years later with onset of painful mandibular tumor recurrence associated with recurrent bilateral lung metastases. He was rechallenged with dabrafenib/trametinib and experienced another prompt tumor response and remains in a second durable clinical remission (currently > 16 months) on continuous dual targeted therapy. We discuss the implications of this case study for future treatment strategies.

BCL-XL PROTAC degrader DT2216 synergizes with sotorasib in preclinical models of KRASG12C-mutated cancers.

KRAS mutations are the most common oncogenic drivers. Sotorasib (AMG510), a covalent inhibitor of KRASG12C, was recently approved for the treatment of KRASG12C-mutated non-small cell lung cancer (NSCLC). However, the efficacy of sotorasib and other KRASG12C inhibitors is limited by intrinsic resistance in colorectal cancer (CRC) and by the rapid emergence of acquired resistance in all treated tumors. Therefore, there is an urgent need to develop novel combination therapies to overcome sotorasib resistance and to maximize its efficacy. We assessed the effect of sotorasib alone or in combination with DT2216 (a clinical-stage BCL-XL proteolysis targeting chimera [PROTAC]) on KRASG12C-mutated NSCLC, CRC and pancreatic cancer (PC) cell lines using MTS cell viability, colony formation and Annexin-V/PI apoptosis assays. Furthermore, the therapeutic efficacy of sotorasib alone and in combination with DT2216 was evaluated in vivo using different tumor xenograft models. We observed heterogeneous responses to sotorasib alone, whereas its combination with DT2216 strongly inhibited viability of KRASG12C tumor cell lines that partially responded to sotorasib treatment. Mechanistically, sotorasib treatment led to stabilization of BIM and co-treatment with DT2216 inhibited sotorasib-induced BCL-XL/BIM interaction leading to enhanced apoptosis in KRASG12C tumor cell lines. Furthermore, DT2216 co-treatment significantly improved the antitumor efficacy of sotorasib in vivo. Collectively, our findings suggest that due to cytostatic activity, the efficacy of sotorasib is limited, and therefore, its combination with a pro-apoptotic agent, i.e., DT2216, shows synergistic responses and can potentially overcome resistance.

Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial.

Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown activity in melanoma, but has not been previously evaluated in metastatic non-small cell lung cancer. We conducted a single-arm open-label phase 1 trial ( NCT03215810 ) of TILs administered with nivolumab in 20 patients with advanced non-small cell lung cancer following initial progression on nivolumab monotherapy. The primary end point was safety and secondary end points included objective response rate, duration of response and T cell persistence. Autologous TILs were expanded ex vivo from minced tumors cultured with interleukin-2. Patients received cyclophosphamide and fludarabine lymphodepletion, TIL infusion and interleukin-2, followed by maintenance nivolumab. The end point of safety was met according to the prespecified criteria of ≤17% rate of severe toxicity (95% confidence interval, 3-29%). Of 13 evaluable patients, 3 had confirmed responses and 11 had reduction in tumor burden, with a median best change of 35%. Two patients achieved complete responses that were ongoing 1.5 years later. In exploratory analyses, we found T cells recognizing multiple types of cancer mutations were detected after TIL treatment and were enriched in responding patients. Neoantigen-reactive T cell clonotypes increased and persisted in peripheral blood after treatment. Cell therapy with autologous TILs is generally safe and clinically active and may constitute a new treatment strategy in metastatic lung cancer.

Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation.

Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here, we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of human and murine LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.

The CRTC1-MAML2 fusion is the major oncogenic driver in mucoepidermoid carcinoma.

No effective systemic treatment is available for patients with unresectable, recurrent, or metastatic mucoepidermoid carcinoma (MEC), the most common salivary gland malignancy. MEC is frequently associated with a t(11;19)(q14-21;p12-13) translocation that creates a CRTC1-MAML2 fusion gene. The CRTC1-MAML2 fusion exhibited transforming activity in vitro; however, whether it serves as an oncogenic driver for MEC establishment and maintenance in vivo remains unknown. Here, we show that doxycycline-induced CRTC1-MAML2 knockdown blocked the growth of established MEC xenografts, validating CRTC1-MAML2 as a therapeutic target. We further generated a conditional transgenic mouse model and observed that Cre-induced CRTC1-MAML2 expression caused 100% penetrant formation of salivary gland tumors resembling histological and molecular characteristics of human MEC. Molecular analysis of MEC tumors revealed altered p16-CDK4/6-RB pathway activity as a potential cooperating event in promoting CRTC1-MAML2-induced tumorigenesis. Cotargeting of aberrant p16-CDK4/6-RB signaling and CRTC1-MAML2 fusion-activated AREG/EGFR signaling with the respective CDK4/6 inhibitor Palbociclib and EGFR inhibitor Erlotinib produced enhanced antitumor responses in vitro and in vivo. Collectively, this study provides direct evidence for CRTC1-MAML2 as a key driver for MEC development and maintenance and identifies a potentially novel combination therapy with FDA-approved EGFR and CDK4/6 inhibitors as a potential viable strategy for patients with MEC.

Targeting Notch and EGFR signaling in human mucoepidermoid carcinoma.

Mucoepidermoid carcinoma (MEC) is the most common type of salivary gland cancers and patients with advanced, metastatic, and recurrent MECs have limited therapeutic options and poor treatment outcomes. MEC is commonly associated with a chromosomal translocation t(11;19) (q14-21;p12-13) that encodes the CRTC1-MAML2 oncogenic fusion. The CRTC1-MAML2 fusion is required for MEC growth in part through inducing autocrine AREG-EGFR signaling. Growing evidence suggests that MEC malignancy is maintained by cancer stem-like cells. In this study, we aimed to determine critical signaling for maintaining MEC stem-like cells and the effect of combined targeting of stem cell signaling and CRTC1-MAML2-induced EGFR signaling on blocking MEC growth. First, we evaluated the significance of Notch signaling in regulating MEC stem-like cells. Aberrantly activated Notch signaling was detected in human fusion-positive MEC cells. The inhibition of Notch signaling with genetic or pharmacological inhibitors reduced oncosphere formation and ALDH-bright population in vitro and blocked the growth of MEC xenografts in vivo. Next, we investigated the effect of co-targeting Notch signaling and EGFR signaling, and observed enhanced inhibition on MEC growth in vivo. Collectively, this study identified a critical role of Notch signaling in maintaining MEC stem-like cells and tumor growth, and revealed a novel approach of co-targeting Notch and EGFR signaling as a potential effective anti-MEC treatment.

Delta-Like Protein 3 Expression and Targeting in Merkel Cell Carcinoma.

PURPOSE: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker for DLL3-targeting antibody-drug conjugate and other therapies. Given the neuroendocrine features of Merkel cell carcinoma (MCC), we sought to evaluate DLL3 expression and its role in MCC. EXPERIMENTAL DESIGN: Formalin-fixed and paraffin-embedded MCC cases were consecutively selected. Immunohistochemistry was performed for DLL3 (SC16.65 antibody) and polyomavirus large T-antigen (sc-136172 antibody). Slides were read out for percentage of positive tumor cells. Cox proportional hazards model was applied to assess the association between DLL3 expression and overall survival (OS). A patient with a DLL3-expressing MCC was treated with rovalpituzumab tesirine (Rova-T) in the "other tumor" cohort of NCT02709889 and assessed for response. RESULTS: The median H-score of DLL3 expression of 65 patients included was 60 (interquartile range, 30-100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range, 25%-70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = .003) when it was dichotomized to negative versus positive. H-score of DLL3 expression did not predict OS of patients with MCC (p = .4) after being adjusted for common clinicopathological factors. A patient treated with Rova-T for refractory metastatic MCC achieved partial response. CONCLUSIONS: DLL3 overexpression is very common in MCC by immunohistochemistry. The response to treatment suggests that DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. IMPLICATIONS FOR PRACTICE: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify patients for treatment with DLL3-targeting agents. Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. It was found that DLL3 overexpression is very common in MCC by immunohistochemistry and significantly associated with Merkel cell polyomavirus expression. Despite the lack of prognostic significance in this cohort, DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive DLL3-targeting therapies.

Co-occurring Alterations in the RAS-MAPK Pathway Limit Response to MET Inhibitor Treatment in MET Exon 14 Skipping Mutation-Positive Lung Cancer.

PURPOSE: Although patients with advanced-stage non-small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood. EXPERIMENTAL DESIGN: Targeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC. RESULTS: Prominent co-occurring RAS-MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib. CONCLUSIONS: Our study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

MYB-activated models for testing therapeutic agents in adenoid cystic carcinoma.

OBJECTIVE: There are no effective systemic therapies for adenoid cystic cancer (ACC) and lack of tumor lines and mouse models have hindered drug development.We aim to develop MYB-activated models for testing new therapeutic agents. MATERIALS AND METHODS: We studied new ACC patient-derived xenograft (PDX) models and generated a matched cell line from one patient. In addition, we generated a genetically-engineered MYB-NFIB mouse model (GEMM) that was crossed with Ink4a+/-/Arf+/- mice to study tumor spectrum and obtain tumor lines. Using human and murine ACC-like tumor lines, we analyzed MYB expression by RNA-Seq and immunoblot and tested efficacy of new MYB inhibitors. RESULTS: We detected MYB-NFIB transcripts in both UFH1 and UFH2 PDX and observed tumor inhibition by MYB depletion using shRNA in vivo. We observed rapid loss of MYB expression when we cultured UFH1 in vitro, but were able to generate a UFH2 tumor cell line that retained MYB expression for 6 months. RNA-Seq expression detected an ACC-like mRNA signature in PDX samples and we confirmed an identical KMT2A/MLL variant in UFH2 PDX, matched cell line, and primary biopsy. Although the predominant phenotype of the MYB-NFIB GEMM was B-cell leukemia, we also generated a MYB-activated ACC-like mammary tumor cell line. We observed tumor inhibition using a novel MYB peptidomimetic in both human and murine tumor models. CONCLUSIONS: We generated and studied new murine and human MYB-activated tumor samples and detected growth inhibition with MYB peptidomimetics. These data provide tools to define treatment strategies for patients with advanced MYB-activated ACC.