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Supplementary Fig. S2: Spectra of EMT score and AXL expression across different tumor types.

Table S1. Mutations in key oncogenes in lung cancer cell lines tested.

AXL, a TAM (TYRO3, AXL, and MERTK) family receptor tyrosine kinase, is increasingly being recognized as a key determinant of resistance to targeted therapies, as well as chemotherapy and radiation in non–small cell lung cancer (NSCLC) and other cancers. We further show here that high levels of AXL and epithelial-to-mesenchymal transition were frequently expressed in subsets of both treatment-naïve and treatment-relapsed NSCLC. Previously, we and others have demonstrated a role for AXL in mediating DNA damage response (DDR), as well as resistance to inhibition of WEE1, a replication stress response kinase. Here, we show that BGB324 (bemcentinib), a selective small-molecule AXL inhibitor, caused DNA damage and induced replication stress, indicated by ATR/CHK1 phosphorylation, more significantly in TP53-deficient NSCLC cell lines. Similar effects were also observed in large-cell neuroendocrine carcinoma (LCNEC) cell lines. High AXL protein levels were also associated with resistance to ATR inhibition. Combined inhibition of AXL and ATR significantly decreased cell proliferation of NSCLC and LCNEC cell lines. Mechanistically, combined inhibition of AXL and ATR significantly increased RPA32 hyperphosphorylation and DNA double-strand breaks and induced markers of mitotic catastrophe. Notably, NSCLC cell lines with low levels of SLFN11, a known predictive biomarker for platinum and PARP inhibitor sensitivity, were more sensitive to AXL/ATR cotargeting. These findings demonstrate a novel and unexpected role for AXL in replication stress tolerance, with potential therapeutic implications.Implications:These findings demonstrate that the combination of AXL and ATR inhibitors could be a promising therapeutic combination for NSCLC, LCNEC, and other cancers.

Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo, LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. Cancer Res; 77(14); 3870–84. ©2017 AACR.

This file contains additional details of materials and methods used in this study like SCLC cell line authentication, targeted knockdown of CHK1 and MYC, PCR, cell viability aasay, cell cycle analysis, generation of tumor models, RPPA and immunofluorescence.

This file contains supplementary tables Table S1: Genotypic status of human SCLC cell lines and their sensitivity to LY2606368 Table S2: Pairwise p-value of the syngeneic SCLC in vivo survival data of Figure 3 Table S3: Primers sequence for CHEK1 and GAPDH

This file contains the supplementary figures and figure legends. Figure S1: CHK1 overexpression and LY2606368 targeting efficacy in SCLC. Figure S2:Combinatorial effect of LY2606368 and cisplatin. Figure S3: Anti-tumor efficacy of LY2606368. Figure S4: Effect of LY2606368 in platinum-resistant SCLC models. Figure S5: Combinatorial effect of LY2606368 and olaparib. Figure S6 and S7: Proteomic biomarkers of LY2606368 response. Figure S8: MYC expression predicts sensitivity to LY2606368.

BACKGROUND: Lurbinectedin recently received FDA accelerated approval as a second line treatment option for metastatic small cell lung cancer (SCLC). However, there are currently no established biomarkers to predict SCLC sensitivity or resistance to lurbinectedin or preclinical studies to guide rational combinations. METHODS: Drug sensitivity was assayed in proliferation assays and xenograft models. Baseline proteomic profiling was performed by reverse-phase protein array. Lurbinectedin-induced changes in intracellular signaling pathways were assayed by Western blot. RESULTS: Among 21 human SCLC cell lines, cytotoxicity was observed following lurbinectedin treatment at a low dose (median IC50 0.46 nM, range, 0.06–1.83 nM). Notably, cell lines with high expression of Schlafen-11 (SLFN11) protein, a promising biomarker of response to other DNA damaging agents (e.g., chemotherapy, PARP inhibitors), were more sensitive to single-agent lurbinectedin (FC =3.2, P=0.005). SLFN11 was validated as a biomarker of sensitivity to lurbinectedin using siRNA knockdown and in xenografts representing SLFN11 high and low SCLC. Replication stress and DNA damage markers (e.g., γH2AX, phosphorylated CHK1, phosphorylated RPA32) increased in SCLC cell lines following treatment with lurbinectedin. Lurbinectedin also induced PD-L1 expression via cGAS-STING pathway activation. Finally, the combination of lurbinectedin with the ataxia telangiectasia and Rad3-related protein (ATR) inhibitors ceralasertib and berzosertib showed a greater than additive effect in SLFN11-low models. CONCLUSIONS: Together our data confirm the activity of lurbinectedin across a large cohort of SCLC models and identify SLFN11 as a top candidate biomarker for lurbinectedin sensitivity. In SLFN11-low SCLC cell lines which are relatively resistance to lurbinectedin, the addition of an ATR inhibitor to lurbinectedin re-sensitized otherwise resistant cells, confirming previous observations that SLFN11 is a master regulator of DNA damage response independent of ATR, and the absence of SLFN11 leads to synthetic lethality with ATR inhibition. This study provides a rationale for lurbinectedin in combination with ATR inhibitors to overcome resistance in SCLC with low SLFN11 expression.

DNA-damaging agents exploit increased genomic instability, a hallmark of cancer. Recently, inhibitors targeting the DNA damage response (DDR) pathways, such as PARP inhibitors, have also shown promising therapeutic potential. However, not all tumors respond well to these treatments, suggesting additional determinants of response are required. Schlafen 11 (SLFN11), a putative DNA/RNA helicase that induces irreversible replication block, is emerging as an important regulator of cellular response to DNA damage. Preclinical and emerging clinical trial data suggest that SLFN11 is a predictive biomarker of response to a wide range of therapeutics that cause DNA damage including platinum salts and topoisomerase I/II inhibitors, as well as PARP inhibitors, which has raised exciting possibilities for its clinical application. In this article, we review the function, prevalence, and clinical testing of SLFN11 in tumor biopsy samples and circulating tumor cells. We discuss mounting evidence of SLFN11 as a key predictive biomarker for a wide range of cancer therapeutics and as a prognostic marker across several cancer types. Furthermore, we discuss emerging areas of investigation such as epigenetic reactivation of SLFN11 and its role in activating immune response. We then provide perspectives on open questions and future directions in studying this important biomarker.

Objectives: Small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas (NECs) share many clinical features, including limited response to immune checkpoint inhibitors (ICIs). However, preclinical data suggest synergistic response with combined ICI and poly (ADP-ribose) polymerase inhibitors (PARPi) in SCLC models. This single-institution, phase II trial (NCT04701307) assessed the efficacy of a combination of the anti-PD-1 monoclonal antibody, dostarlimab, with the selective PARPi, niraparib, in patients with relapsed SCLC or NECs. Methods: Eligible patients with SCLC (Cohort 1) or NECs (Cohort 2) had received at least one prior line of therapy and were treated with niraparib 300 mg (or 200 mg if Results: From Feb 2021 to Aug 2021, 14 and 9 patients enrolled with SCLC and NEC, respectively. NECs included tumors from gynecological, head/neck, and gastrointestinal sites. In Cohort 1 (SCLC), 12 of 14 patients were evaluable by RECISTv1.1, including 1 complete response (CR), while 4 patients not achieving partial response (PR) had minor responses (-2%, -14%, -19%, and -24% from baseline). Only 1 patient in this cohort achieved PFS6, though 2 additional patients progressed between 5 and 6 months. In Cohort 2 (NEC), 6 of 9 patients were evaluable by RECISTv1.1 and no CR/PRs were observed. One patient did experience durable minor response (-21%), but no patients achieved PFS6. Dose-limiting, niraparib-related hematological AEs, typically thrombocytopenia, occurred in 12 of 23 patients. Non-hematological, treatment-related serious AEs occurred in only 1 patient (Grade 3 inflammatory arthritis), while 1 additional patient experienced brief dose-interruption due to Grade 2 pneumonitis. Preliminary translational data, including bulk and single-cell RNAseq, indicate molecular features, such as transcriptional subtype, are shared across SCLC and NECs and may predict response to this combination. Conclusions: In heavily pre-treated patients across SCLC and NEC cohorts, combined niraparib and dostarlimab failed to exceed interim futility criteria. However, multiple SCLC patients experienced durable disease control, including one patient with sustained CR and associated translational studies point to potential biomarker-driven approaches in the future. Trial supported by GlaxoSmithKline. Citation Format: Carl M. Gay, C. Allison Stewart, Michael Frumovitz, Junya Fujimoto, Yuann xi, Qi Wang, Runsheng Wang, Veronica Novegil, Mehmet Altan, Tina Cascone, Arvind Dasari, Yasir Y. Elamin, Frank V. Fossella, Bonnie S. Glisson, Charles S. Lu, Marcelo V. Negrao, Ferdinandos Skoulidis, Natalie Vokes, Robert J. Cardnell, Ignacio I. Wistuba, Jing Wang, John V. Heymach, Lauren A. Byers. A phase II trial of niraparib plus dostarlimab in relapsed small cell lung cancer and other high-grade neuroendocrine carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT218.

Neuroendocrine carcinomas (NECs) are clinically aggressive carcinomas commonly arising from the respiratory and gastrointestinal tracts, typically categorized as large-cell neuroendocrine carcinomas (LCNECs) or small cell carcinomas (most commonly small cell lung cancer (SCLC)). Clinically, pulmonary LCNECs (pLCNECs) mirror the course common to SCLC - initial response followed by rapid and insurmountable resistance to one-size-fits-all approaches. Recently, SCLC has been subdivided into four subtypes with unique vulnerabilities, three of which are defined by the transcription factors ASCL1, NEUROD1, and POU2F3, while a fourth group exhibits an inflamed signature. We hypothesize that pLCNEC may be similarly classified into molecularly distinct subsets with unique therapeutic vulnerabilities - a fundamental step toward personalized medicine. We applied our SCLC 1300 gene signature to pLCNEC patient tumors and, as in SCLC, found three distinct subtypes determined by differential expression of ASCL1, NEUROD1, and POU2F3, but with a unique fourth subtype marked by expression of the transcription factor YAP1. Unlike in treatment-naïve SCLC, where YAP1 is absent, YAP1 expression clearly defines pLCNEC as two, roughly equal subsets with the YAP1-low tumors encompassing tumors expressing the other three transcription factors. Conversely, YAP1-high pLCNEC is more mesenchymal and inflamed, and less neuroendocrine (NE), reminiscent of inflamed SCLC. Additionally, YAP1-high status is associated with smoking exposure (P Citation Format: C. Allison Stewart, Lixia Diao, Yuanxin Xi, Runsheng Wang, Kavya Ramkumar, B. Leticia Rodriguez, Benjamin B. Morris, Li Shen, Bingnan Zhang, Yan Yang, Azusa Tanimoto, Veronica Y. Novegil, Luisa M. Solis Soto, Pedro F. Simoes da Rocha, Natalie Vokes, Don L. Gibbons, Michael Frumovitz, Junya Fujimoto, Jing Wang, Bonnie Glisson, Lauren A. Byers, Carl M. Gay. YAP1 in relapsed pulmonary high-grade neuroendocrine carcinomas (NEC) is associated with CDKN2A loss, intact RB1, EMT and therapeutic vulnerability to MEK1 and CDK4/6 inhibition. [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 4525.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor. Despite high initial responses to frontline chemo-immunotherapy, therapeutic resistance develops rapidly. There are limited treatment options in the relapsed setting, where the prognosis remains dismal. SCLC tumors experience continuous and high levels of replication stress (RS) due to ubiquitous loss of key cell cycle checkpoints, RB1 and TP53. Frequent amplification and high expression of the transcription factor cMYC further contribute to increased RS. Thus, high levels of RS expose a potential SCLC vulnerability and provide a therapeutic opportunity. Our group and others have shown that AXL, a TAM family receptor tyrosine kinase that is highly expressed in mesenchymal tumors, mediates resistance to chemotherapy, radiation and targeted therapies in SCLC, non-small cell lung cancer and other cancers, through its role in driving epithelial to mesenchymal transition (EMT). More recently, a novel role for AXL in DNA damage repair and tolerance has emerged. Therefore, we hypothesize that AXL targeting may be a potential therapeutic approach in SCLC. We first investigated the transcriptomic expression profile of AXL in SCLC clinical cohorts. AXL-high tumors were seen in a subset of treatment-naïve SCLC tumors, frequently among, but not limited to, the inflamed SCLC subtype. AXL expression was also seen in many relapsed SCLC tumors. As expected, tumors with high AXL expression also expressed several mesenchymal genes and higher EMT scores. Interestingly, among the treatment-naïve SCLC tumors, AXL expression was inversely correlated with a RS signature (rho=-0.54, p Citation Format: Kavya Ramkumar, C. Allison Stewart, Azusa Tanimoto, Qi Wang, Yuanxin Xi, Benjamin B. Morris, Runsheng Wang, Li Shen, Robert J. Cardnell, Jing Wang, Carl M. Gay, Lauren A. Byers. Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer [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 6206.

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By age 20 weeks, uterine gland development was comparable to controls, but pathologies, including cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue postpubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in postpubertal uterine gland development. However, progesterone treatment did not rescue postpubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts postpubertal uterine gland development and homeostasis.

Introduction Despite the enthusiasm surrounding cancer immunotherapy, most SCLC patients show very modest response to immune checkpoint inhibitor monotherapy treatment. Therefore, there is growing interest in combining immune checkpoint blockade with chemotherapy and other treatments to enhance immune checkpoint blockade efficacy. Based on favorable clinical trial results, chemotherapy and immunotherapy combinations have been recently approved by the U.S. Food and Drug Administration for frontline treatment for SCLC. Methods and Results Here, we show that combined treatment of SRA737, an oral CHK1 inhibitor, and anti–programmed death ligand 1 (PD-L1) leads to an antitumor response in multiple cancer models, including SCLC. We further show that combining low, non-cytotoxic doses of gemcitabine with SRA737 + anti–PD-L1/anti–PD-1 significantly increased antitumorigenic CD8+ cytotoxic T cells, dendritic cells, and M1 macrophage populations in an SCLC model. This regimen also led to a significant decrease in immunosuppressive M2 macrophage and myeloid-derived suppressor cell populations, as well as an increase in the expression of the type I interferon beta 1 gene, IFNβ, and chemokines, CCL5 and CXCL10. Conclusions Given that anti–PD-L1/anti–PD-1 drugs have recently been approved as monotherapy and in combination with chemotherapy for the treatment of SCLC, and that the SRA737 + low dose gemcitabine regimen is currently in clinical trials for SCLC and other malignancies, our preclinical data provide a strong rational for combining this regimen with inhibitors of the PD-L1/PD-1 pathway.

Background: SARS-CoV-2 infection is the cause of the respiratory illness COVID-19, which presents most frequently with respiratory symptoms SARS-CoV-2 cell entry requires interactions with ACE2 and TMPRSS2 on the surface of the host cell Cancer patients and, specifically, those with thoracic malignancies seem to experience poorer clinical outcomes Methods: We utilized bulk and single-cell transcriptional data from a combination of normal and malignant tissues and cells from aerodigestive and respiratory tracts to explore mechanisms governing the expression of ACE2 and TMPRSS2 Additionally, we determined the effect of EMT induction, ZEB1 modulation, and SARS-CoV-2 infection on ACE2 expression Results: Our bulk data suggests that aerodigestive and lung cancer models express a broad range of ACE2 and TMRPSS2, particularly in epithelial cells, and would serve as good models for studying SARS-CoV-2 infection We assessed the relationship between ACE2 and epithelial differentiation in numerous datasets, and found consistent positive correlations with transcriptional and microRNA signifiers of epithelial differentiation The miR-200 family – zinc finger E-box-binding homeobox 1 (ZEB1) pathway, which is an established regulator of EMT, also directly regulates ACE2 expression, likely via putative ZEB1 repressor sites located in the ACE2 promoter Furthermore, SARS-CoV-2 infection reduces ACE2 expression and shifts cells to a more mesenchymal phenotype with loss of EPCAM and upregulation of ZEB1 and other EMT-associated genes Conclusions: ACE2-positive cells are almost exclusively epithelial and unexpectedly rare, considering the devastating impact of this infection Following viral entry, SARS-CoV-2 infection induces molecular changes within the cells that are reminiscent of EMT, including increased ZEB1 ZEB1, in turn, appears to directly repress the expression of ACE2 This SARS-CoV-2-induced ACE2 deficiency, compounded by the downregulation of genes, including claudins, which play a critical role in restricting epithelial and endothelial permeability, exposes respiratory cells to increased risk of edema and acute respiratory distress syndrome (ARDS) Legal entity responsible for the study: The authors Funding: NIH/NCI R01-CA207295 (L A B ), NIH/NCI U01-CA213273 (L A B , J V H ), CCSG P30-CA01667 (L A B ), University of Texas SPORE in Lung Cancer P5-CA070907 (L A B , D L G , J V H , C M G ), the Department of Defense (LC170171;L A B ), Khalifa Bin Zayed Al Nahyan Foundation (C M G ), RP170067 (EMP), through generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shot Program and Andrew Sabin Family Fellowship, and The Rexanna Foundation for Fighting Lung Cancer Disclosure: C Gay: Research grant/Funding (self): Astra Zeneca J V Heymach: Advisory/Consultancy: AstraZeneca;Advisory/Consultancy: Boehringer Ingelheim;Advisory/Consultancy: Exelixis;Advisory/Consultancy: Genentech;Advisory/Consultancy: GlaxoSmithKline;Advisory/Consultancy: Guardant Health;Advisory/Consultancy: Hengrui;Advisory/Consultancy: Spectrum L A Byers: Advisory/Consultancy, Research grant/Funding (self): AstraZeneca;Advisory/Consultancy, Research grant/Funding (self): AbbVie;Advisory/Consultancy, Research grant/Funding (self): GenMab;Advisory/Consultancy: BergenBio;Advisory/Consultancy: Pharma Mar SA;Advisory/Consultancy, Research grant/Funding (self): Sierra Oncology;Advisory/Consultancy: Merck;Advisory/Consultancy: Bristol Myers Squibb;Advisory/Consultancy: Genentech;Advisory/Consultancy: Pfizer;Research grant/Funding (self): Tolero Pharmaceuticals All other authors have declared no conflicts of interest

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by robust, however transient, responses to frontline platinum-based therapy that are rapidly followed by refractory relapses. Treatment resistance is a significant concern in SCLC and a major factor regulating resistance is an emergence of unique cellular populations and an increase in intratumoral heterogeneity (ITH). We hypothesize that these emerging populations expressing various resistance-associated pathways, represent multiple, unique drug-resistant persister cell populations that underlie increased ITH and promote therapeutic resistance and, eventually, relapse, despite the initial response to therapy. Persister cells represent a unique population of cancer cells that are resistant to therapeutic pressure. While most persister cells remain arrested under treatment, a rare sub-population exists that can reverse state and re-enter the cell cycle. We performed single cell RNAseq of relapsed, extensive stage SCLC patient paired core biopsies collected 1) following relapse to standard of care (SOC) platinum chemotherapy plus immune checkpoint blockade (ICB) and 2) after six weeks of further therapy. Unlike what we found in platinum-sensitive disease, there is not a meaningful change in ITH score between cancer cells in the first and second biopsy. Presumably, maximum heterogeneity developed along with relapse to SOC treatment. Biopsies represent SCLC across subtypes (SCLC-A/N/P/I) with a modest loss of transcription factor expression following treatment (e.g., 76.8% to 57.9% NEUROD1). Cancer cells were classified as cycling or non-cycling to identify potential persister cell populations in paired patient core biopsies. Molecular subtype marker ASCL1 was reduced in non-cycling cells, but there was no change in NEUROD1 or POU2F3. Cycling cells demonstrate increased expression of NE genes (SYP, INSM1, UCHL1), biomarkers of response (SLFN11 and CDH1), but also genes associated with resistance (EZH2, NFIB). Non-cycling cells exhibit some common resistance mechanisms (e.g., EMT and MYC, AXL, ZFP36L1, and REST overexpression) and increased expression of inflammatory genes (e.g. HLA family) compared to cycling cells, reminiscent of the SCLC-Inflamed (SCLC-I) subtype. These data suggest that non-cycling persister cells may be more sensitive to ICB, AXL and/or AURK inhibition, while cycling cells may be more responsive to platinum chemotherapy, epigenetic modifiers or DNA repair targeted therapies. Clinically, these data underscore the importance of maximizing and maintaining the initial response in platinum-sensitive SCLC tumors, while highlighting the transcriptional complexity underlying SCLC’s profound treatment resistance following SOC and address the major need to develop combination therapies to target these distinct cell populations. Citation Format: C. Allison Stewart, Yuanxin Xi, Runsheng Wang, Michael M. Frumovitz, Jing Wang, Lauren A. Byers, Carl M. Gay. Defining the transcriptional complexity of persister cell populations in relapsed small cell lung cancer patient biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1598.

The natural history of small-cell lung cancer (SCLC) includes rapid evolution from chemosensitivity to chemoresistance, although mechanisms underlying this evolution remain obscure due to the scarcity of post-relapse tissue samples. We generated circulating tumor cell (CTC)-derived xenografts from patients with SCLC to study intratumoral heterogeneity (ITH) via single-cell RNA sequencing of chemosensitive and chemoresistant CTC-derived xenografts and patient CTCs. We found globally increased ITH, including heterogeneous expression of therapeutic targets and potential resistance pathways, such as epithelial-to-mesenchymal transition, between cellular subpopulations following treatment resistance. Similarly, serial profiling of patient CTCs directly from blood confirmed increased ITH post-relapse. These findings suggest that treatment resistance in SCLC is characterized by coexisting subpopulations of cells with heterogeneous gene expression leading to multiple, concurrent resistance mechanisms. These findings emphasize the need for clinical efforts to focus on rational combination therapies for treatment-naive SCLC tumors to maximize initial responses and counteract the emergence of ITH and diverse resistance mechanisms. Stewart et al. use circulating tumor cell-derived xenografts from patients with small-cell lung cancer to study tumor heterogeneity following the onset of therapeutic resistance.

MYC stimulates both metabolism and protein synthesis, but how cells coordinate these complementary programs is unknown. Previous work reported that, in a subset of small-cell lung cancer (SCLC) cell lines, MYC activates guanosine triphosphate (GTP) synthesis and results in sensitivity to inhibitors of the GTP synthesis enzyme inosine monophosphate dehydrogenase (IMPDH). Here, we demonstrated that primary MYC(hi) human SCLC tumors also contained abundant guanosine nucleotides. We also found that elevated MYC in SCLCs with acquired chemoresistance rendered these otherwise recalcitrant tumors dependent on IMPDH. Unexpectedly, our data indicated that IMPDH linked the metabolic and protein synthesis outputs of oncogenic MYC. Coexpression analysis placed IMPDH within the MYC-driven ribosome program, and GTP depletion prevented RNA polymerase I (Pol I) from localizing to ribosomal DNA. Furthermore, the GTPases GPN1 and GPN3 were upregulated by MYC and directed Pol I to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigated the effect of GTP depletion on Pol I, protecting chemoresistant SCLC cells from IMPDH inhibition. GTP therefore functioned as a metabolic gate tethering MYC-dependent ribosome biogenesis to nucleotide sufficiency through GPN1 and GPN3. IMPDH dependence is a targetable vulnerability in chemoresistant MYC(hi) SCLC.

Despite molecular and clinical heterogeneity, small cell lung cancer (SCLC) is treated as a single entity with predictably poor results. Using tumor expression data and non-negative matrix factorization, we identify four SCLC subtypes defined largely by differential expression of transcription factors ASCL1, NEUROD1, and POU2F3 or low expression of all three transcription factor signatures accompanied by an Inflamed gene signature (SCLC-A, N, P, and I, respectively). SCLC-I experiences the greatest benefit from the addition of immunotherapy to chemotherapy, while the other subtypes each have distinct vulnerabilities, including to inhibitors of PARP, Aurora kinases, or BCL-2. Cisplatin treatment of SCLC-A patient-derived xenografts induces intratumoral shifts toward SCLC-I, supporting subtype switching as a mechanism of acquired platinum resistance. We propose that matching baseline tumor subtype to therapy, as well as manipulating subtype switching on therapy, may enhance depth and duration of response for SCLC patients. Gay et al. provide a classification for four subtypes of small cell lung cancer, each with unique molecular features and therapeutic vulnerabilities. An inflamed, mesenchymal subtype predicts benefit with the addition of immunotherapy to chemotherapy. Intratumoral switching between chemosensitive and chemoresistant subtypes accompanies therapeutic resistance.

AXL, a TAM (TYRO3, AXL, and MERTK) family receptor tyrosine kinase, is increasingly being recognized as a key determinant of resistance to targeted therapies, as well as chemotherapy and radiation in non–small cell lung cancer (NSCLC) and other cancers. We further show here that high levels of AXL and epithelial-to-mesenchymal transition were frequently expressed in subsets of both treatment-naïve and treatment-relapsed NSCLC. Previously, we and others have demonstrated a role for AXL in mediating DNA damage response (DDR), as well as resistance to inhibition of WEE1, a replication stress response kinase. Here, we show that BGB324 (bemcentinib), a selective small-molecule AXL inhibitor, caused DNA damage and induced replication stress, indicated by ATR/CHK1 phosphorylation, more significantly in TP53-deficient NSCLC cell lines. Similar effects were also observed in large-cell neuroendocrine carcinoma (LCNEC) cell lines. High AXL protein levels were also associated with resistance to ATR inhibition. Combined inhibition of AXL and ATR significantly decreased cell proliferation of NSCLC and LCNEC cell lines. Mechanistically, combined inhibition of AXL and ATR significantly increased RPA32 hyperphosphorylation and DNA double-strand breaks and induced markers of mitotic catastrophe. Notably, NSCLC cell lines with low levels of SLFN11, a known predictive biomarker for platinum and PARP inhibitor sensitivity, were more sensitive to AXL/ATR cotargeting. These findings demonstrate a novel and unexpected role for AXL in replication stress tolerance, with potential therapeutic implications. Implications: These findings demonstrate that the combination of AXL and ATR inhibitors could be a promising therapeutic combination for NSCLC, LCNEC, and other cancers.

Background Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine malignancy that accounts for 15% of lung cancer diagnoses. The severity of this disease is exacerbated by the fact that there are few therapeutic options, which mostly offer limited clinical benefit, culminating in a 5-year survival rate of Methods To identify transcriptional subtypes, we used non-negative matrix factorization of gene expression data from 81 SCLC tumors and identified four subtypes largely based on differential expression of the transcription factors ASCL1, NEUROD1, and POU2F3. We hypothesized that these subtypes may underlie unique therapeutic vulnerabilities. We examined differential expression of genes that encode surface-expressed proteins that may be targetable by reagents such as therapeutic antibodies or antibody-drug conjugates (ADCs). Results Our four subtypes are defined either by high expression of ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P), or an absence of those transcription factors and instead a prevalence of immunological factors (SCLC-Inflamed, or SCLC-I). We curated a list of approximately 60 candidate genes encoding surface proteins that are differentially expressed across the four subtypes. Within these 60 candidates, we have identified a few specific to each subtype for which there exist clinically available, targeted ADCs. The most prevalent subtype, SCLC-A, showed high expression of targets such as DLL3 (SCLC-A) and CEACAM5 (SCLC-A). SCLC-N highly expressed SSTR2, a somatostatin receptor that is being actively targeted in SCLC clinical trials. The two non-neuroendocrine subtypes, SCLC-P and SCLC-I shared some common hits such as the NK cell ligand MICA and B7H6. All of the identified and highlighted hits have been or are actively being pursued in clinical trials, highlighting the importance of understanding their expression levels pre- and post-treatment so that novel therapies can be developed that will be effective over the course of disease progression Conclusions The underlying biology defining our four identified subtypes of SCLC has revealed a striking number of targetable, differentially expressed surface protein encoding genes many of which already have clinically available reagents that could be repurposed for treatment of SCLC on a subtype-specific basis.

Endometrial or uterine glands secrete substances essential for uterine receptivity to the embryo, implantation, conceptus survival, and growth. Adenogenesis is the process of gland formation within the stroma of the uterus. In the mouse, uterine gland formation initiates at postnatal day (P) 5. Uterine gland morphology is poorly understood because it is primarily based on two-dimensional (2D) histology. To more fully describe uterine gland morphogenesis, we generated three-dimensional (3D) models of postnatal uterine glands from P0 to P21, based on volumetric imaging using light sheet microscopy. At birth (P0), there were no glands. At P8, we found bud- and teardrop-shaped epithelial invaginations. By P11, the forming glands were elongated epithelial tubes. By P21, the elongated tubes had a sinuous morphology. These morphologies are homogeneously distributed along the anterior–posterior axis of the uterus. To facilitate uterine gland analyses, we propose a novel 3D staging system of uterine gland morphology during development in the prepubertal mouse. We define five uterine gland stages: Stage 1: bud; Stage 2: teardrop; Stage 3: elongated; Stage 4: sinuous; and Stage 5: primary branches. This staging system provides a standardized key to assess and quantify prepubertal uterine gland morphology that can be used for studies of uterine gland development and pathology. In addition, our studies suggest that gland formation initiation occurs during P8 and P11. However, between P11 and P21 gland formation initiation stops and all glands elongate and become sinuous. We also found that the mesometrial epithelium develops a unique morphology we term the uterine rail.

Small cell lung cancer (SCLC) is an aggressive form of neuroendocrine carcinoma, notable for early metastases and rapid relapse despite initial response to frontline platinum-based chemotherapy. To date, there are no validated predictive biomarkers in SCLC, hence all patients are treated the same way. Preclinical studies identified SLFN11, a putative DNA/RNA helicase that blocks replication at stressed replication fork, as a predictive biomarker to a wide range of agents targeting DNA damage such as platinum, topoisomerase I/II inhibitors and PARP inhibitors. Based on these observations, pre-specified biomarker analyses in a clinical trial demonstrated SLFN11 predicts better clinical outcomes in SCLC patients when treated with PARP inhibitor combinations such as temozolomide and veliparib. To better characterize the prevalence, heterogeneity and predictive value of SLFN11 in SCLC, we developed and validated a SLFN11 immunohistochemistry (IHC) assay meeting Clinical Laboratory Improvements Amendments (CLIA) standards, and a novel circulating tumor cell (CTC) assay (Epic Sciences®) to detect the expression level of SLFN11 in SCLC tumors or CTCs and correlated with clinical outcomes. We found that SLFN11 was expressed by IHC in roughly 50% of the SCLC clinical tumor samples, from three separate clinical trial cohorts (total of 207 extensive-stage SCLC patient samples). There was a wide range of H-scores by IHC which suggests heterogeneity in SLFN11 expression (H-score range 1.5-235). Similarly, analyses of patient CTCs from blood samples confirmed that SLFN11 is expressed in about 50% of treatment-naïve patients, however SLFN11 expression decreased significantly in patients on platinum treatment and at the time of relapse. Most patients had CTCs with pathologic features consistent with SCLC (i.e., were small, round, had high nuclear-to-cytoplasm ratios, and had salt-and-pepper like chromatin textures), although inter- and intra- patient heterogeneity was observed and SLFN11 expression was observed independent of morphologic subtype. Interestingly, SLFN11 expression was also found in white blood cells in the blood samples and was highest in platinum-naïve patients and lowest in patients while on platinum. Together, these data highlight the potential of SLFN11 as a predictive biomarker in SCLC. Based on our group and others' previous work, SLFN11 positivity by IHC is being used for selection of patients in an ongoing clinical trial (NCT04334941). In addition, given the substantial challenge of obtaining adequate tumor tissue in SCLC either at initial diagnosis or with re-biopsies, blood-based CTC analyses are an important tool to detect SLFN11 expression. Because of the dynamic nature of SLFN11 expression, CTC analyses can be especially valuable for longitudinal monitoring and may have real-time implications for treatment choice and response. Citation Format: Bingnan Zhang, C. Allison Stewart, Carl M. Gay, Qi Wang, Robert Cardnell, Junya Fujimoto, Luisa Fernandez, Adam Jendrisak, Cole Gilbertson, Joseph Schonhoft, Joshua Jones, Amanda K. Anderson, Ignacio I. Wistuba, Jing Wang, Rick Wenstrup, Lauren A. Byers. Detection of DNA replication blocker SLFN11 in tumor tissue and circulating tumor cells to predict platinum response in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 384.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung malignancy which is primarily driven by loss of function of tumor suppressor gene TP53 and RB1 and accounts for around 13-15% of all lung cancers. For metastatic SCLC the standard-of-care, first-line therapy is combination of platinum-based therapy with etoposide or with irinotecan. Though response rate in first-line chemotherapy in SCLC is very high, relapse is almost universal. For decades topotecan, a topoisomerase-I inhibitor was the only FDA approved second-line treatment in SCLC. However, on June 15th, 2020 the FDA approved lurbinectedin for treatment of patients with metastatic SCLC with disease progression from platinum-based chemotherapy and designated it as an orphan drug. Lurbinectedin (PM01183) is a synthetic analog of the natural marine-based tetrahydroisoquinoline, trabectedin which comes from the sea-squirt species Ecteinascidia turbinate. Lurbinectedin blocks transcription by inhibiting the activity of RNA-polymerase-II and inducing its specific degradation by the ubiquitin/proteasome machinery, also inducing DNA damage. Emerging data from our group, and others supports that SCLC is transcriptionally addicted via one of three main transcription factors ASCL1 (A), NeuroD1 (N) & POU2F3 (P), which may contribute to the promising results observed in SCLC trials to date with lurbinectedin. However, there are currently no established biomarkers to predict SCLC sensitivity or resistance to lurbinectedin. Furthermore, little is known regarding molecular changes in SCLC cells or other tumors upon exposure to lurbinectedin. In this preclinical study we investigated the therapeutic efficacy of lurbinectedin in large number of profiled SCLC cell lines representing the four SCLC subtypes (A, N, P and I) to identify candidate markers of drug sensitivity and resistance. In 12 human-derived and 3 mouse model-derived SCLC cell lines, the majority of cell lines were highly sensitive to lurbinectedin at a very low doses (median IC50 0.52 nM, range 0.08-1.84nM). We also observed increased markers of DNA damage following treatment in sensitive cell lines (e.g., γH2AX, ChK1, RPA32) by western blot. Notably, no subtype specific difference in lurbinectedin sensitivity was observed among the four subtypes (A, N, P and I) of SCLC cells. However, cell lines with higher SLFN11 expression were more sensitive to lurbinectedin. Western blot experiments showed significant increase in phosphorylation of γH2AX, ChK1, RPA32 in lurbinectedin treated SCLC cells compared to DMSO treatment. Together our preliminary data confirm lurbinectedin as a potent treatment option for SCLC, and support a rationale for potential combinations with other DNA damaging agents. Citation Format: Kiran Kundu, Robert J. Cardnell, Li Shen, C. Allison Stewart, Kasey Cargill, Carl M. Gay, Jing Wang, Lauren A. Byers. Characterization of lurbinectedin as a single agent and in combinations with DNA damage response inhibitor for the treatment and bio-marker discovery of SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1022.

Background Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy with dismal survival outcomes and no established predictive biomarkers. The landmark randomized, phase III IMpower133 trial established the new frontline standard of care for extensive-stage SCLC (ES-SCLC) as etoposide/platinum (EP) plus immune checkpoint blockade (ICB) [anti-PD-L1; atezolizumab (atezo)] based on an overall survival (OS) benefit compared to EP plus placebo. However, this survival benefit is limited in unselected populations, emphasizing the need for predictive biomarkers. Preclinically, there is emerging evidence of transcriptional heterogeneity among SCLC tumors, but the impact on therapeutic benefit remains undefined. Using non-negative matrix factorization (NMF) analysis of gene expression data from 81 SCLC tumors samples, we previously identified four subtypes, including three defined largely by differential expression of the transcription factors ASCL1 (SCLC-A), NEUROD1 (SCLC-N), and POU2F3 (SCLC-P), and a novel, fourth subtype with low expression of all three transcription factor signatures. Method and Results Using transcriptional and proteomic data from patient tumors and tumor-derived models, we molecularly characterized each of the four identified subtypes. The previously undescribed fourth subtype, dubbed SCLC-Inflamed (SCLC-I) showed high expression of non-neuroendocrine transcription factors (e.g. REST) and markers of EMT. Most distinctly, relative to the “cold” immune microenvironment typical of SCLC tumors, SCLC-I tumors possess markedly higher expression of interferon-γ signatures and immune checkpoints, including CD274 (PD-L1). Furthermore, cell type deconvolution using CIBERSORTx identified significantly higher infiltration into SCLC-I tumors by multiple immune cell types including T-cells, NK cells, macrophages, and dendritic cells. We predicted SCLC-I might derive disproportionate benefit from ICB due to its inflamed features. To test this, we applied our NMF-derived gene signature to 276 treatment-naïve, ES-SCLC patient tumors from the IMpower133 trial to assign patient subtype. The distribution of subtypes was as follows: SCLC-A 51%, SCLC-N 23%, SCLC-I 18% and SCLC-P 7%. While there was a trend toward OS benefit with the addition of atezo in each subtype, the benefit was numerically greater in SCLC-I. Specifically, median OS (atezo vs placebo arm) in months (mo) was 18.2 mo vs 10.4 mo for SCLC-I tumors, while median OS for the other three subtypes ranged from 9.6-10.9 mo (atezo arm) and 6.0-10.6 mo (placebo arm). Conclusion Unbiased transcriptional analyses identify four subtypes with distinct tumor and immune features. While all subtypes experienced improved OS with addition of anti-PD-L1 to frontline EP, SCLC-I patients appear to experience the most durable benefit. Citation Format: Carl M. Gay, C. Allison Stewart, Lixia Diao, Barzin Y. Nabet, Junya Fujimoto, Luisa M. Solis, Wei Lu, Yuanxin Xi, Robert J. Cardnell, Natalie I. Vokes, Kavya Ramkumar, Stephen G. Swisher, Jack A. Roth, Bonnie S. Glisson, David S. Shames, Ignacio I. Wistuba, Jing Wang, John Minna, John V. Heymach, Lauren A. Byers. A novel, inflamed small cell lung cancer transcriptional subtype, SCLC-I, defines a subset of patients with distinct immunotherapy vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 22.

Small cell lung cancer (SCLC) accounts for approximately 15% of lung cancer cases with a poor five year survival rate of 6%. The National Cancer Institute has designated SCLC as one of two “recalcitrant” cancers and urges translational research to advance treatment options. In our effort to maximize treatment benefits, we have aimed our investigation at uncovering metabolic differences contributing to disease progression that may be targeted through specific pathway inhibition. Although metabolism is relatively unstudied in SCLC, metabolic reprogramming is recognized as a hallmark of cancer and can be regulated by several different mechanisms. Notably, the transcription factor MYC is overexpressed in 30% SCLC tumors and is known to modulate the balance between two major pathways of metabolism: glycolysis and mitochondrial respiration. While mitochondrial respiration is the preferred source of energy production in terminally differentiated cells, many cancer cells switch to become predominantly glycolytic in an effort to generate energy quickly and produce biomolecules and electron carriers required for proliferation—a phenomena known as the Warburg Effect. Since MYC is expressed in only a subset of highly aggressive SCLC, we applied bimodal separation to both patient and cell line SCLC datasets to establish defined MYC subsets (MYCLow; MYCHigh) and performed gene ontology pathway analysis to genes that were significantly upregulated among the MYC-expressing samples. This revealed that 37% of significantly upregulated genes were linked to metabolic processes and further investigation showed many of those genes were linked to the glycolysis pathway. We also confirmed upregulation of protein expression through reverse phase protein array (RPPA). With glycolysis proving to be increased in MYCHigh samples, we simultaneously characterized pathway utilization in cell lines at baseline and in the presence of a potent glycolysis inhibitor. MYCHigh cell lines exhibited greater glucose consumption and lactate secretion, which was significantly decreased by glycolytic inhibition. Likewise, mitochondrial analysis revealed lower oxygen consumption and ATP production that were further reduced in the presence of the glycolysis inhibitor. Although mitochondrial density was unchanged regardless of MYC expression or glycolytic inhibition, reactive oxygen species (ROS) generation was greatly enhanced in the MYCHigh subset upon suppression of glycolysis. Lastly, SCLC xenografts derived from a MYCHigh SCLC cell line showed significantly slower tumor growth whereas MYCLow derived xenografts exhibited no significant difference in tumor growth. Together, these data provide evidence of metabolic differences among SCLC subsets such that MYC expression induces reliance on glycolysis, which can be targeted for therapeutic intervention. Citation Format: Kasey R. Cargill, C. Allison Stewart, Elizabeth M. Park, Robert J. Cardnell, You Hong Fan, Qi Wang, Lixia Diao, Wai Kin Chan, Philip L. Lorenzi, Jing Wang, Lauren A. Byers. Targeting MYC-enhanced glycolysis in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2335.

Introduction: Despite the recent approval of immune checkpoints inhibitors (ICI) as a treatment option in the extensive-stage small cell lung cancer (SCLC) setting, survival has not significantly changed in the last decades. Recent scientific efforts have led to the identification of 4 major subtypes defined by expression of three transcription factors: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) with the fourth subtype characterized by increased expression of immune genes (Inflamed subtype - SCLC-I). Transcriptomic results, along with recent immunotherapy trials, suggest that modulation of tumor immune microenvironment (TIME) could potential be critical to achieving clinical responses in a subset of patients, hence a comprehensive study of the TIME in SCLC is imperative. Here we report the feasibility of a multiplex immunofluorescence (mIF) methodology to analyze the TIME in SCLC. Methods: FFPE sections from surgically resected SCLC (N=4, one representative case across all SCLC subtypes) were identified from the ICON Project at UT MD Anderson Cancer Center. We used mIF to identify and quantify immune markers grouped into two 6-antibody panels: Panel 1: cytokeratin (CK, AE1/AE3), CD3, CD8, PD-1, PD-L1 and CD68; Panel 2: CK, CD20, granzyme B, FOXP3, CD45RO, and CD57. Finally, genomic (WES), transcriptomic (RNA sequencing) and proteomic (RPPA) data from these cases were integrated with the mIF data. Results: SCLC molecular subtypes (SCLC-A, N, P, I) were classified using transcriptomic and proteomic data. Analysis of TIME unveils a higher immune cell infiltration within SCLC-I subtype compared with the other cases representing, immune “cold” SCLC subtypes. SCLC-I subtype showed a 2-13 folder higher (range) immune cell density than SCLC-A, N and P subtypes (measured as a median of cell density). Specifically, T cells (CD3+) (695 and 242 cells/mm2, for SCLC-I and the median for the other subtypes respectively), T cytotoxic cells (CD3+ CD8+) (206 and 105), activated T cells (CD3+ CD8+ granzyme+) (20 and 2), antigen experienced ‘like' T cells (CD3+ PD-1+) (17 and 0), memory T cells (CD3+ CD45RO+) (328 and 91) and macrophages (CD68+) (773 and 57). PD-L1 expression in malignant cells did not show significant differences within the 4 SCLC subtypes. However, PD-L1 expression in macrophages was significantly higher in the SCLC-I subtype, suggesting an increase of IFN-gamma in the TIME. Conclusions: TIME study show the use of mIF in SCLC tumors to be feasible, and could potentially provide key information towards the identification of SCLC patients that could benefit from ICI. For the first time we complemented transcriptomic data from SCLC tumors with mIF analysis unveiling the complex interplay of the host immune response and malignant cells. Our preliminary results warrant further studies to explore the role of TIME in immunotherapeutic response in SCLC. Citation Format: Pedro Rocha, C. Allison Stewart, Edwin Parra, Luisa M. Solis, Carl M. Gay, Robert J. Cardnell, Naohiro Uraoka, Alejandro Francisco-Cruz, Hitoshi Dejima, Yuanxin Xi, Lixia Diao, Jing Wang, Marcelo V. Negrao, Jianjun Zhang, Ignacio Wistuba, Don L. Gibbons, Lauren A. Byers. Multiplex immunofluorescence (mIF) reveals differences in tumor immune microenvironment between molecularly-defined subsets of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2758.

// C. Allison Stewart 1 , Pan Tong 2 , Robert J. Cardnell 1 , Triparna Sen 1 , Lerong Li 2 , Carl M. Gay 1 , Fatemah Masrorpour 1 , You Fan 1 , Rasha O. Bara 1 , Ying Feng 3 , Yuanbin Ru 3 , Junya Fujimoto 4 , Samrat T. Kundu 1 , Leonard E. Post 3 , Karen Yu 3 , Yuqiao Shen 3 , Bonnie S. Glisson 1 , Ignacio Wistuba 4 , John V. Heymach 1 , Don L. Gibbons 1 , Jing Wang 2 and Lauren Averett Byers 1 1 Department of Thoracic Head & Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 3 BioMarin Pharmaceutical, San Rafael, CA 94901, USA 4 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Correspondence to: Lauren Averett Byers, email: lbyers@mdanderson.org Keywords: SCLC, SLFN11, ATM, EMT, PARP inhibitor Received: December 10, 2016 Accepted: January 19, 2017 Published: February 15, 2017 ABSTRACT Small cell lung cancer (SCLC) is one of the most aggressive forms of cancer, with a 5-year survival

Introduction Although the combination of anti–programmed cell death-1 or anti–programmed cell death ligand-1 (PD-L1) with platinum chemotherapy is a standard of care for NSCLC, clinical responses vary. Even though predictive biomarkers (which include PD-L1 expression, tumor mutational burden, and inflamed immune microenvironment) are validated for immunotherapy, their relevance to chemoimmunotherapy combinations is less clear. We have recently reported that activation of the stimulator of interferon genes (STING) innate immune pathway enhances immunotherapy response in SCLC. Here, we hypothesize that STING pathway activation may predict and underlie predictive correlates of antitumor immunity in NSCLC. Methods We analyzed transcriptomic and proteomic profiles in two NSCLC cohorts from our institution (treatment-naive patients in the Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax study and relapsed patients in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination study) and The Cancer Genome Atlas (N = 1320). Tumors were stratified by STING activation on the basis of protein or mRNA expression of cyclic GMP-AMP synthase, phospho-STING, and STING-mediated chemokines (chemokine ligand 5 [CCL5] and C-X-C motif chemokine 10 [CXCL10]). STING activation in patient tumors and in platinum-treated preclinical NSCLC models was correlated with biomarkers of immunotherapy response. Results STING activation is associated with higher levels of intrinsic DNA damage, targetable immune checkpoints, and chemokines in treatment-naive and relapsed lung adenocarcinoma. We observed that tumors with lower STING and immune gene expression show higher frequency of serine-threonine kinase 11 (STK11) mutations; however, we identified a subset of these tumors that are TP53 comutated and display high immune- and STING-related gene expression. Treatment with cisplatin increases STING pathway activation and PD-L1 expression in multiple NSCLC preclinical models, including adeno- and squamous cell carcinoma. Conclusions STING pathway activation in NSCLC predicts features of immunotherapy response and is enhanced by cisplatin treatment. This suggests a possible predictive biomarker and mechanism for improved response to chemoimmunotherapy combinations.

Small cell lung cancer (SCLC), the most aggressive form of lung cancer, is notable for early dissemination and impressive, yet transient, responses to frontline chemotherapy that are rapidly undone by refractory relapses. To gain a better understanding of resistance mechanisms, we generated circulating tumor cell-derived xenograft (CDX) models from liquid biopsies of SCLC patients and treated them with chemotherapy or targeted agents. As expected, CDXs demonstrate similar chemotherapy response to the patient from whom they were derived. We have previously established that baseline chemosensitive CDXs are molecularly homogeneous at the single cell level, while chemoresistant CDXs exhibit increased intratumoral heterogeneity (ITH) with distinct variations in gene expression between cancer cell populations. We hypothesize that paired analyses of chemosensitive CDXs will demonstrate increased ITH after developing acquired resistance and are likely to offer more specific insights into resistance mechanisms than prior work with unrelated CDX models. Platinum-sensitive CDXs were treated with cisplatin or DNA damage response targeted therapies (PARP inhibitor or CHK inhibitor) continuously until tumor progression was observed. To identify transcriptional changes associated with onset of resistance, single-cell RNAseq analysis was performed on vehicle-treated and relapsed CDX tumors. We found globally increased ITH including heterogeneous expression of therapeutic targets and potential resistance pathways, such as EMT, between cellular subpopulations following treatment-resistance. Relapse was consistently associated with increased ITH score (P Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Santhosh Sivajothi, Junya Fujimoto, Patrice M. Hartsfield, Hai Tran, Stephen G. Swisher, Jack A. Roth, Jianjun Zhang, Bonnie Glisson, John V. Heymach, Ignacio Wistuba, Paul Robson, Jing Wang, Lauren A. Byers. Paired, single-cell profiling of circulating tumor cell-derived xenograft models of small cell lung cancer reveals intratumoral heterogeneity and emergence of new cell clusters following treatment relapse [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1496.

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By 20 weeks of age, uterine gland development was comparable to controls, but pathologies, including squamous neoplasia, tubal neoplasia, and cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue post-pubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in post-pubertal uterine gland development. However, progesterone treatment did not rescue post-pubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts post-pubertal uterine gland development and homeostasis.

Endometrial or uterine glands secrete substances essential for uterine receptivity to the embryo, implantation, conceptus survival, development, and growth. Adenogenesis is the process of gland formation within the stroma of the uterus that occurs after birth. In the mouse, uterine gland formation initiates at postnatal day (P) 5. Subsequently, the developing uterine glands invade into the adjacent stroma. Mouse uterine gland morphology is poorly understood because it is based on two-dimensional (2D) histological observations. To more fully describe uterine gland morphogenesis, we generated three-dimensional (3D) models of postnatal uterine glands from P0 to P21, using light sheet microscopy. At birth (P0), there were no glands. At P8, we found bud- and teardrop-shaped epithelial invaginations. By P11, the forming glands were elongated epithelial tubes. By P21, the elongated tubes had a sinuous morphology. These morphologies are homogeneously distributed along the anterior-posterior axis of the uterus. To facilitate uterine gland analyses, we propose a novel 3D staging system of uterine gland morphology during postnatal development in the mouse. We define 6 stages: Stage 0: Aglandular, Stage 1: Bud, Stage 2: Teardrop, Stage 3: Elongated, Stage 4: Sinuous, and Stage 5: Primary Branches. This staging system provides a standardized key to assess and quantify uterine gland morphology that can be used for studies of uterine gland development and pathology. In addition, our studies suggest that gland formation initiation occurs during P8 and P11. However, between P11 and P21 gland formation initiation stops and all glands elongate and become sinuous.

Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo , LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. Cancer Res; 77(14); 3870–84. ©2017 AACR .

Accounting for 15% of all lung cancer diagnoses, small cell lung cancer (SCLC) is an aggressive malignancy with dismal clinical outcomes, due in part to failure to define SCLC molecular subsets and identify the unique, targetable vulnerabilities therein. Recent data has begun to delineate these subsets by uncovering inter-tumoral heterogeneity in features such as DNA damage response, EMT, and neuroendocrine (NE) status. An integrated analysis of clinical samples to determine the implications of this heterogeneity broadly on SCLC classification has not yet been performed. Using RNAseq data from 81 SCLC tumor samples, we applied non-negative matrix factorization (NMF) which identified three clusters, each enriched for unique transcriptional programs driven by ASCL1 (30/81), NEUROD1 (24/81), or POU2F3 (27/81). These three genes encode transcription factors which define mutually exclusive NE-high, NE-low, and novel tuft cell variants of SCLC. Bulk RNAseq analyses of SCLC CTC-derived xenograft (CDX) models validated the clustering analysis in vivo. However, single-cell RNAseq from these same models reveals subtle evidence of intra-tumoral and intra-cellular heterogeneity in transcriptional programs that appear mutually exclusive in bulk analyses, suggesting plasticity among these variants. Guided by our NMF results, we performed RNAseq-based supervised clustering to classify each of 60 SCLC cell lines into ASCL1-driven, NEUROD1-driven, and POU2F3-driven clusters. Then, using reverse phase protein array data for these lines, we derived proteomic signatures for each cluster as follows: ASCL1-driven: E-cadherinhigh/TTF-1high/cMYClow, NEUROD1-driven: E-cadherinlow/TTF-1low/cMYChigh, and POU2F3-driven: E-cadherinhigh/TTF-1low/cMYChigh (ANOVA p Citation Format: Carl M. Gay, Lixia Diao, C. Allison Stewart, Yuanxin Xi, Robert J. Cardnell, Stephen G. Swisher, Jack A. Roth, Bonnie S. Glisson, Jing Wang, John V. Heymach, Lauren A. Byers. Inter- and intra-tumoral variations in ASCL1, NEUROD1, and POU2F3 transcriptional programs underlie three distinct molecular subtypes of small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3772.

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by rapid onset of platinum-resistance. However, mechanisms underlying platinum-resistance remain obscure due to scarcity of tissue samples from relapsed patients. We generated circulating tumor cell (CTC)-derived xenograft (CDX) models from SCLC patients that recapitulate patient tumor genomics and response to platinum chemotherapy. Little is known about whether intratumoral heterogeneity (ITH) exists in SCLC and how it may contribute to clinical outcomes and development of treatment resistance. To investigate this, we performed baseline single-cell RNAseq analyses of platinum-sensitive and -resistant CDX models, as well as longitudinal single-cell RNAseq analyses of CDX models and patient CTCs over the course of therapy. Within each CDX model, we observe not only increased ITH with resistance (variance-based metric, P=0.018), but distinct cellular populations with unique gene signatures associated with resistance (e.g. EMT, DNA damage repair, MYC activation, etc.). To confirm this relationship between ITH and resistance, platinum-sensitive CDX models were subjected to extended treatment with DNA damage response targeted therapies until relapse occurred. Single-cell RNAseq confirmed that, as predicted, untreated tumors were molecularly homogeneous, while relapse was associated with increased ITH and multiple, concurrent mechanisms of resistance, including TGF beta signaling and G2/M checkpoints. Unexpectedly, we found variations in the mechanisms of resistance within replicate treatment-relapsed mice, suggesting that resistance even to molecularly targeted therapies does not follow a predictable, reproducible pathway. For example, onset of resistance to a PARP inhibitor resulted in upregulation of NOTCH signaling in one tumor, but not others. Similarly, longitudinal single-cell profiling of CTCs directly from patient blood before, during, and after platinum-relapse confirmed increased ITH post-relapse accompanying unique mechanisms of resistance within specific cell populations (e.g., MYC activation, EMT, and TNFα signaling). We independently found ITH of protein expression (e.g., SLFN11, EZH2, EMT) in CTCs isolated from patient blood, signifying a method for measuring ITH clinically. These data suggest that treatment resistance in SCLC entails a fluid process of shifting expression profiles to generate an increasingly heterogeneous tumor with multiple, disparate mechanisms of resistance. Clinically, these findings imply that drug development efforts in this disease should focus on combination or maintenance therapies for treatment-naïve SCLC tumors to maximize depth of initial responses and delay the onset of resistance defined by ITH. Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Junya Fujimoto, Neda Kalhor, Patrice M. Hartsfield, Hai Tran, Luisa Fernandez, David Lu, Yipeng Wang, Ryan Dittamore, Jianjun Zhang, Stephen G. Swisher, Jack A. Roth, Trudy G. Oliver, John V. Heymach, Ignacio I. Wistuba, Bonnie S. Glisson, Paul Robson, Jing Wang, Lauren A. Byers. Single-cell analyses reveal increasing intratumoral heterogeneity as an essential component of treatment resistance in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2899.

Müllerian duct differentiation and development into the female reproductive tract is essential for fertility, but mechanisms regulating these processes are poorly understood. WNT signaling is critical for proper development of the female reproductive tract as evident by the phenotypes of Wnt4, Wnt5a, Wnt7a, and β-catenin (Ctnnb1) mutant mice. Here we extend these findings by determining the effects of constitutive CTNNB1 activation within the mesenchyme of the developing Müllerian duct and its differentiated derivatives. This was accomplished by crossing Amhr2-Cre knock-in mice with Ctnnb1 exon (ex) 3(f/f) mice. Amhr2-Cre(Δ/+); Ctnnb1 ex3(f/+) females did not form an oviduct, had smaller uteri, endometrial gland defects, and were infertile. At the cellular level, stabilization of CTNNB1 in the mesenchyme caused alterations within the epithelium, including less proliferation, delayed uterine gland formation, and induction of an epithelial-mesenchymal transition (EMT) event. This EMT event is observed before birth and is complete within 5 days after birth. Misexpression of estrogen receptor α in the epithelia correlated with the EMT before birth, but not after. These studies indicate that regulated CTNNB1 in mesenchyme is important for epithelial cell differentiation during female reproductive tract development.

Uterine gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition. Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition. endometrium, female reproductive tract, uterine gland, uterus

Small cell lung cancer (SCLC) is an aggressive form of lung cancer, notable for rapid emergence of drug resistance following initial chemotherapy. Rates of five-year overall survival are only 7% across all stages and only one drug, topotecan, is approved by the FDA for recurrent SCLC. As a result, the National Cancer Institute has named identifying novel vulnerabilities in SCLC as an urgent area of need. Increased expression, relative to non-small cell lung cancer (NSCLC), of numerous components of the DNA damage response (DDR) pathway, including poly (ADP-Ribose) polymerase 1 (PARP1) and ataxia telangiectasia mutated (ATM), is observed in SCLC. Thus, targeting DDR has emerged as an attractive therapeutic strategy in SCLC, bolstered by recent data demonstrating activity of PARP1 inhibitors (PARPi) in SCLC patients. Interestingly, data suggest that PARPi resistant models from other tumors may rely on another DDR component, ataxia telangiectasia and Rad3 related protein (ATR), for survival. ATR/ATR is also highly expressed in SCLC compared to NSCLC and normal lung tissue. Preclinical data have shown that treatment with ATR inhibitors (ATRi) is especially effective in p53- and ATM-deficient tumor models, a notable fact given that SCLC is universally p53-mutant and that ATM-mutant and ATM-deficient SCLC is a small, but significant proportion of all SCLC. We treated 22 human-derived SCLC cell line models with two clinically relevant ATRi, VX-970 (formerly VE-822) and AZD-6738, and observed single agent activity of both ATR inhibitors in a significant number of cell lines, with half-maximal inhibitory concentrations (IC50s) as low as 30 nM and >100-fold difference in IC50s between the most and least sensitive cell lines. Utilizing extensive genomic, transcriptomic and proteomic characterization of these cell lines, we then identified predictive biomarkers of response to ATRi in SCLC, including low ATM expression. As low ATM was associated with ATRi sensitivity, we tested whether the addition of an ATM inhibitor (AZD-0156) may further sensitize SCLC models to ATRi. We treated 12 SCLC cell lines with AZD-6738 and AZD-0156 in combination and identified a subset of lines in which synergy is observed between the two agents. Similarly, as targeting ATR has been shown to overcome PARPi resistance in other cancer types, we treated 12 SCLC cell lines with the ATRi AZD-6738 and the PARPi olaparib in combination and again observed a subset of lines in which the two agents acted synergistically. Interestingly, the lines in which ATRi+ATMi and ATRi+PARPi synergy is observed are distinct and include lines that were the most resistant to single-agent AZD-6738. Together, these data support further investigation of ATRi in SCLC and suggest that via the use of ATRi alone or in combination with ATMi or PARPi, multiple molecularly distinct subsets of SCLC can be effectively targeted. Citation Format: Carl M. Gay, Pan Tong, Lerong Li, C. Allison Stewart, Triparna Sen, Bonnie S. Glisson, John V. Heymach, Jing Wang, Lauren Averett Byers. ATR inhibitors are active as single agents and in combination with PARP1 and ATM inhibitors in molecularly distinct subsets of small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2822.

Small cell lung cancer (SCLC) accounts for 14% of lung cancer diagnoses in the United States and is characterized by rapid onset of chemoresistance and poor clinical outcomes. Once considered a homogeneous disease, recent analyses of SCLC have identified intratumoral heterogeneity (ITH) with respect to NOTCH signaling, ASCL1/NEUROD1 balance and MYC amplification - all of which are potential mechanisms underlying SCLC's aggressive and refractory biology. Unfortunately, patient-derived models of SCLC with which to better characterize the molecular profiles of refractory SCLC are scarce. To address this, we generated circulating tumor cell-derived xenograft (CDX) models from liquid biopsies of patients with treatment-naïve or relapsed SCLC. Each CDX model underwent pathological review to confirm tumors were consistent with SCLC based on histology and standard immunohistochemical markers (e.g., TTF1, chromogranin A, synaptophysin, NCAM). Sequencing of these models revealed mutations typical of SCLC (e.g. TP53, RB1), which were maintained in vivo over multiple passages. Importantly, each model's in vivo response to cisplatin matched the patient's platinum response at the time of CDX generation. At the proteomic level, platinum-resistant models exhibited mTOR activation, increased SOX2 and ATM, and reduced E-cadherin, suggesting a shift toward EMT and cancer stem cell expansion may contribute to resistance. To investigate ITH, we analyzed single-cell gene expression profiles by RNAseq using a droplet-based Chromium Single Cell system that analyzed a filtered subsample of 2000 cells per tumor. Consistent with SCLC, all CDX models contained large numbers of cells expressing neuroendocrine-specific genes (SYP, CHGA). However, Principle Component Analysis revealed that cells from chemosensitive CDX models had distinct expression profiles from resistant models. Using our published EMT gene signature, we found that resistant models had higher proportions of mesenchymal (vs. epithelial) cells. Several other distinctions between sensitive and resistant models were detected at the single-cell level but not in bulk RNA and protein analyses, suggesting that single-cell resolution can identify occult platinum-resistant subpopulations. For example, higher proportions of ASCL1- and DLL3-expressing cells were associated with platinum sensitivity, whereas a shift toward predominant NEUROD1-expression was observed with resistance. Cells expressing each of these three genes were identified across all tumors, suggesting platinum-sensitive and resistant subpopulations are ubiquitous but that even subtle shifts in the fractional distribution of these subsets can exert significant impact on response. These data support further use of single-cell analysis to explore the role of ITH as a driver of drug resistance in SCLC. Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Siva V., Junya Fujimoto, Pan Tong, Lixia Diao, Lerong Li, Mohan Bolisetty, Neda Kalhor, Patrice Lawson, Mayra Vasquez, Hai Tran, Ignacio I. Wistuba, Bonnie Glisson, Jianjun Zhang, Stephen G. Swisher, Jack A. Roth, John V. Heymach, Paul Robson, Jing Wang, Lauren A. Byers. Single-cell profiling of small cell lung cancer circulating tumor cell-derived xenograft models reveals intratumoral heterogeneity among mediators of chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 990.

In this issue of Cancer Cell, Mohammad et al. describe LSD1, a histone demethylase, as a therapeutic target in SCLC with a unique epigenetic signature to predict drug sensitivity. Inhibition of LSD1 reduces cell proliferation and stem cell maintenance while promoting cell differentiation and reducing tumor growth in preclinical models.

The understanding of the reproductive events and the molecular mechanisms regulating fertility and infertility in humans relies heavily on the analysis of the corresponding phenotypes in mouse models. While molecular genetic approaches provide significant insight into the molecular regulation of these processes, the lack of live imaging methods that allow for detailed visualization of the mouse reproductive organs limits our investigations of dynamic events taking place during the ovulation, the fertilization and the pre-implantation stages of embryonic development. Here we introduce an in vivo three-dimensional imaging approach for visualizing the mouse oviduct and reproductive events with micro-scale spatial resolution using optical coherence tomography (OCT). This method relies on the natural tissue optical contrast and does not require the application of any contrast agents. For the first time, we present live high-resolution images of the internal structural features of the oviduct, as well as other reproductive organs and the oocytes surrounded by cumulus cells. These results provide the basis for a wide range of live dynamic studies focused on understanding fertility and infertility.

Background: WEE1 inhibitors (WEEi) are a novel class of small molecule inhibitors that are now under early clinical trials for small cell lung cancer (SCLC). WEE1 is a protein kinase that plays a key role in regulating the G2 checkpoint in response to DNA damage. Moreover, because WEE1 inhibition induces a HR deficient state, dual targeting of WEE1 and PARP (another promising target previously identified in our group) may induce synthetic lethality. In the present study we evaluate the efficacy of AZD1775 alone and with the PARP inhibitor, olaparib; investigate the mechanisms of primary resistance to AZD1775 in SCLC models; and assess the combinatorial efficacy of AZD1775 treatment with the chemotherapy drug, temozolomide (TMZ), that has shown promise in patients with recurrent SCLC and has been included in the National Comprehensive Cancer Network guidelines for standard treatment for SCLC. Results: Combination of AZD1775 with olaparib revealed an additive effect in vitro in 90% of SCLC cell lines. AZD1775 combined with TMZ synergistically decreased viability, increased DNA damage and apoptosis in SCLC cell lines (n=10) irrespective of MGMT status or initial response to AZD1775 alone. However, unlike the sensitive cells, SCLC cell lines showing primary resistance to AZD1775 had an intact DNA repair mechanism (after DNA damage), which may contribute to the resistance mechanism. Proteomic analysis revealed AXL (receptor tyrosine kinase) and phospho-S6K (S240/244) as markers of AZD1775 resistance and treatment with AXL inhibitor, TP0903 (40nM), resensitized the cells to AZD1775. Pre and post-AZD1775 treated samples revealed sustained activation of mTOR pathway in AZD1775 primary resistant lines. We further demonstrated that pre-treatment of the cells with the mTOR inhibitor everolimus sensitized SCLC cells to AZD1775 by causing downregulation of AKT/mTOR pathway. Conclusion: WEE1 inhibitors are currently in clinical trials for SCLC patients. However, as with any targeted therapy, drug resistance is an important barrier to clinical benefit which could be addressed with therapeutic combinations. We show the efficacy of single agent AZD1775 and in combination with olaparib. WEE1 inhibitor, AZD1775 synergizes with TMZ irrespective of MGMT status in all tested in vitro models which warrants further clinical investigation. We also show that the activity of the WEE1 inhibitors might be limited in cancer cells overexpressing of AXL and activated mTOR pathway and that AXL and mTOR inhibition re-sensitized the cells to AZD1775. Our work supports further exploration of the combination of PARP and WEE1 in SCLC and also the possibility of AXL/mTOR inhibition as a mechanism to overcome WEE1 inhibition resistance in SCLC. SCLC is a disease with very limited therapeutic options and no targeted agents with proven benefit; thus the results from this study have clear translational benefit. Citation Format: Triparna Sen, Pan Tong, Lixia Diao, You Hong-Fan, C. Allison Stewart, John V. Heymach, Jing Wang, Lauren A. Byers. WEE1 inhibitor activity correlates to AXL/mTOR expression and exhibits synergy with temozolomide (TMZ) in small cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 298. doi:10.1158/1538-7445.AM2017-298

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.

Small cell lung cancer (SCLC) is an aggressive disease that accounts for 14% of lung cancers. Very little progress has been made towards the treatment of SCLC in the past four decades and there are no established biomarkers to predict effective therapies for patients. In other cancers, SLFN11 plays an important role in sensitizing cancer cells to topoisomerase inhibitors, DNA synthesis inhibitors and alkylating agents. Previously, our lab identified an increase in poly (ADP-Ribose) polymerase 1 (PARP1), an enzyme involved in DNA damage repair, in SCLC patients and cell lines. PARP inhibitors demonstrate significant anti-tumor activity in cell line and animal models of SCLC and are currently being tested in clinical trials for SCLC patients. In Ewing sarcoma (EWS), SLFN11 has been proposed as a biomarker of PARP inhibitor response. Because both EWS and SCLC have high PARP levels and respond favorably to PARP inhibition, we hypothesized that SLFN11 may also be a biomarker for drug sensitivity in SCLC. Using SCLC patient tumors and a large panel of molecularly profiled SCLC cell lines, we investigated the expression of SLFN11 in SCLC and its association with in vitro sensitivity to PARP inhibition (olaparib) and chemotherapy. SLFN11 mRNA levels are significantly higher in SCLC patient tumors relative to normal lung tissue (P = 0.005). In a panel of 51 SCLC cell lines, higher SLFN11 protein expression correlates with both cisplatin (P Citation Format: C. Allison Stewart, Pan Tong, Robert Cardnell, Triparna Sen, Fatemah Mina Masrorpour, Youhong Fan, Jing Wang, Lauren Averett Byers. SLFN11 is a biomarker of sensitivity to PARP inhibition and chemotherapy in small cell lung cancer (SCLC). [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 3870.

The oviduct, or Fallopian tube in humans, transports oocytes and sperm, serves as the site of fertilization, and supports early embryonic development. The oviduct is essential for fertility. In the mouse, the oviduct is a coiled, complex structure that develops from the simple embryonic Müllerian duct. The oviduct consists of four segments, including the infundibulum, ampulla, isthmus, and uterotubal junction. Additionally, the mouse oviduct forms coils, develops longitudinal folds, and undergoes both mesenchymal and epithelial differentiation. Oviduct development and differentiation occurs perinatally. Several signaling pathways have been found to be involved in oviduct formation, such as Wnt, Tgfβ, microRNA processing, as well as others. Overall, the process of oviduct development is poorly understood and can be utilized to further knowledge of epithelial-mesenchymal interactions, regulation of coiling, characteristics of pseudostratified epithelia, and smooth muscle differentiation.

GnRH, produced in the hypothalamus, acts on pituitary gonadotropes to stimulate release of the gonadotropins LH and FSH. Reduced responsiveness of gonadotropes to GnRH is a primary cause of hypogonadotropic hypogonadism (HH), a disease characterized by gonadal dysfunction and low blood levels of gonadotropins. Loss-of-function mutations in the gene encoding the receptor for GnRH (GNRHR) are a common cause of HH. Sequencing of the GNRHR gene in patients with HH revealed mainly point mutations producing single amino acid substitutions that cause misfolding and misrouting of this G protein-coupled receptor. To generate a mouse model that mimics the human disease, we introduced a single amino acid substitution (E90K) into the mouse Gnrhr gene, which is identical to a known human recessive mutation. In humans, E90K causes severe HH by preventing formation of the E90-K121 salt bridge, which is essential for correct folding. In cell cultures, E90K causes misfolding that leads to almost complete retention by the protein quality control system and subsequent degradation. Here we report that the primary phenotype of mice homozygous for E90K is female infertility due to ovulation failure. Mutant males are fertile despite reduced gonadotropin levels and smaller testes. These results suggest decreased GnRH receptor signaling in the mutant animal, compared with wild type. Our findings suggest that a threshold level of GnRH receptor activity is required for ovulation.

The oviduct, or Fallopian tube in humans, transports oocytes and sperm, serves as the site of fertilization, and supports early embryonic development. The oviduct is essential for fertility. In the mouse, the oviduct is a coiled, complex structure that develops from the simple embryonic Mullerian duct. The oviduct consists of four segments, including the infundibulum, ampulla, isthmus, and uterotubal junction. Additionally, the mouse oviduct forms coils, develops longitudinal folds, and undergoes both mesenchymal and epithelial differentiation. Oviduct development and differentiation occurs perinatally. Several signaling pathways have been found to be involved in oviduct formation, such as Wnt, Tgfβ, microRNA processing, as well as others. Overall, the process of oviduct development is poorly understood and can be utilized to further knowledge of epithelial–mesenchymal interactions, regulation of coiling, characteristics of pseudostratified epithelia, and smooth muscle differentiation.