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Purpose: RB1 mutations and loss of retinoblastoma (Rb) expression represent consistent but not entirely invariable hallmarks of small cell lung cancer (SCLC). The prevalence and characteristics of SCLC retaining wild-type Rb are not well-established. Furthermore, the performance of targeted next-generation sequencing (NGS) versus immunohistochemistry for Rb assessment is not well-defined. Experimental Design: A total of 208 clinical SCLC samples were analyzed by comprehensive targeted NGS, covering all exons of RB1, and Rb IHC. On the basis of established coordination of Rb/p16/cyclinD1 expression, p16-high/cyclinD1-low profile was used as a marker of constitutive Rb deficiency. Results: Fourteen of 208 (6%) SCLC expressed wild-type Rb, accompanied by a unique p16-low/cyclinD1-high profile supporting Rb proficiency. Rb-proficient SCLC was associated with neuroendocrine-low phenotype, combined SCLC with non-SCLC (NSCLC) histology and aggressive behavior. These tumors exclusively harbored CCND1 amplification (29%), and were markedly enriched in CDKN2A mutations (50%) and NSCLC-type alterations (KEAP1, STK11, FGFR1). The remaining 194 of 208 SCLC were Rb-deficient (p16-high/cyclinD1-low), including 184 cases with Rb loss (of which 29% lacked detectable RB1 alterations by clinical NGS pipeline), and 10 cases with mutated but expressed Rb. Conclusions: This is the largest study to date to concurrently analyze Rb by NGS and IHC in SCLC, identifying a 6% rate of Rb proficiency. Pathologic-genomic data implicate NSCLC-related progenitors as a putative source of Rb-proficient SCLC. Consistent upstream Rb inactivation via CDKN2A/p16↓ and CCND1/cyclinD1↑ suggests the potential utility of CDK4/6 inhibitors in this aggressive SCLC subset. The study also clarifies technical aspects of Rb status determination in clinical practice, highlighting the limitations of exon-only sequencing for RB1 interrogation. See related commentary by Mahadevan and Sholl, p. 4603

Background: Neuroendocrine lung cancer carcinomas (Lung NECs), which include large cell neuroendocrine carcinomas (LCNECs) and small cell lung cancer (SCLC) tumors, are particularly aggressive lung neoplasms with limited clinical therapeutic options. Patients with lung NECs are treated with platinum-based chemotherapy as the first line of treatment, but they promptly develop resistance. Lurbinectedin has recently been approved by the FDA as a second-line treatment for SCLC due to its efficacy in metastatic chemorelapsed SCLC patients. However, there is still a large percentage of SCLC patients who do not respond to lurbinectedin and the overall survival of those who benefit from it remains very low ( Methods: We performed synergy proliferation assays in vitro. DNA damage/repair pathways were analyzed by western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Results: We explored the cytotoxic capacity of the combination of lurbinectedin with selinexor in cell linesfrom both SCLC (N=2) and LCNECs (N=3) in synergy in vitro assays. Selinexor strongly synergized with lurbinectedin in both SCLC and LCNEC settings and induced a potent increase in the DNA damage marker γ-H2A, reflective of increased DNA damage induced by the combination treatment. Mechanistically, this combination altered DNA damage and DNA repair mechanisms by decreasing CHK1 and CHK2 protein levels in short-term treatments, suggestive of the impairment of DNA response. Additionally, selinexor reduced the expression of MLH, a key regulator of DNA mismatch repair in monotherapy and in combination with lurbinectedin. Consistent with these results, selinexor in combination with lurbinectedin also induced a significant increase in apoptotic cells and promoted cell cycle arrest. Conclusions: These data indicates that inhibition of exportin 1 with selinexor in combination with lurbinectedin is a promising therapeutic strategy in lung NECs. Subsequent In vivo testing in patient-derived xenograft to study efficacy and toxicity of this combination will provide preclinical rationale for clinically exploring this combination in patients with lung NECs who have failed chemotherapy regimen. Citation Format: Esther Redin Resano, Charles Rudin, Alvaro Quintanal-Villalonga. Exportin 1 inhibition synergizes with lurbinectedin by altering the response to DNA damage in neuroendocrine lung tumors. [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 6188.

Lineage plasticity is the ability for a cell to change its phenotypic state under evolutionary pressure. This process mediates acquired resistance to EGFR-targeted therapies in lung adenocarcinoma (LUAD), through transformation to small cell lung cancer (SCLC), an aggressive cancer type of neuroendocrine (NE) histology. This transformed SCLC has worse clinical outcomes than either its LUAD or de novo SCLC counterparts, with less than one-year overall survival following transformation. Classical SCLC itself can display plasticity through interconversion between classical, variant, and non-NE transcriptional subtypes, with non-classical subtypes displaying increased metastasis and chemoresistance. It is poorly understood what factors drive SCLC transformation or subtype switching. Prior studies poorly model intratumoral heterogeneity because they profile tumors in bulk, which only estimates the average phenotype. However, SCLC transformation often results in admixed LUAD/SCLC tumors. We sought to capture the full intratumoral heterogeneity of SCLC transformation and identify potential molecular determinants of plasticity by applying single-cell RNA sequencing (scRNA-seq) to 41 tumors and patient-derived xenografts from 22 patients with transformed or combined LUAD/NE histology, with matched targeted DNA sequencing and 17 de novo SCLC tumors, 10 LUAD tumors with concurrent EGFR/RB1/TP53 mutations, and 4 tumor-adjacent normal lung samples for comparison. Of 234,322 single transcriptomes assessed, we found 89,113 NE cancer cells and 18,197 LUAD cancer cells. We confirmed that NE and LUAD components within the same tumor shared clonal mutations and common ancestry. Compared to de novo, transformed SCLC harbored greater phenotypic diversity across patients (p < 1 × 10−10), driven largely by enrichment of variant and non-NE subtypes (p < 0.02), including NEUROD1, POU2F3, and YAP1-high subtypes, the latter of which was completely absent in de novo. Within each tumor, transformed SCLC displayed higher intratumoral subtype diversity than de novo SCLC (likelihood ratio p < 0.025). After adjusting for SCLC subtype, differential expression and pathway analysis demonstrated that transformed SCLC is a distinct phenotype from de novo and shares features of the ancestral clone that include residual EGFR and NSCLC gene signatures, as well as pathways in neuronal stemness, MYC targets, AKT/MTOR signaling, JAK/STAT inflammation, and chromatin remodeling. In sum, we find increased intratumoral phenotypic diversity in transformed SCLC, including variant and non-NE subtypes, that may explain worse clinical outcomes. We show that transformed SCLC is a distinct phenotype from de novo, marked by pathways that may offer new potential drug targets to constrain plasticity, with the goal of restoring original sensitivity to targeted therapies. Citation Format: Joseph Minhow Chan, Alvaro Quintanal-Villalonga, Amin Sabet, Parvathy Manoj, Ojasvi Chaudhary, Tianhao Xu, Ignas Masilionis, Jacklynn Egger, Noor Sohail, Jaeyoung Chun, Tal Nawy, Linas Mazutis, Triparna Sen, Ronan Chaligne, Helena Yu, Dana Pe'er, Charles Rudin. Single-cell transcriptomic profiling of SCLC transformation reveals increased intratumoral diversity of variant and non-neuroendocrine subtypes [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 1167.

Lineage plasticity, the ability of cells to transdifferentiate between committed developmental pathways, has been proposed as a source of intratumoral heterogeneity and as a mechanism for tumor adaptation to stringent environmental conditions. Lineage plasticity is increasingly recognized as a contributor to both drug resistance and metastasis, as recently highlighted by our team (Quintanal-Villalonga et al., Nat Rev Clin Oncol 2020), thus representing a biological phenomenon with high clinical relevance. Leveraging bulk and single-cell multi-omic approaches in clinical specimens, we aimed to perform a comprehensive molecular characterization of lung cancer cellular plasticity in the settings of (1) histological transdifferentiation as a mechanism of resistance to targeted therapy, and (2) disease progression. In lung adenocarcinomas (LUADs), lineage plasticity drives small cell (SCLC) and squamous cell (LUSC) transdifferentiation in the context of acquired resistance to targeted inhibitors. In the EGFR -mutant cancers, transformation to SCLC and to LUSC as a mechanism of acquired TKI resistance has been reported in 14% and 9% of cases, respectively. Transdifferentiated tumors portend poorer prognosis than non-transformed tumors. To date, no preventative therapies for transformation are available, although tumor subsets at high risk of transformation (concomitant TP53/RB1 inactivation in the case of SCLC transdifferentiation) have been identified. Defining molecular mechanisms of histological transformation in lung cancer has been challenging due in part to a paucity of well-annotated pre- and post-transformation clinical samples. We hypothesized that mixed histology tumors (LUAD/SCLC and LUAD/LUSC), containing different histological components and sharing common driver mutations, may represent an intermediate step of transdifferentiation, and an ideal substrate to study lineage plasticity, with both histological components sharing location, time, and treatment influence. By selecting mixed histology specimens amenable for clean microdissection of each histological component (10 LUAD/LUSC and 11 LUAD/SCLC), as well as pre- and post-transdifferentiated samples (N=17), we performed comprehensive genomic, epigenomic, transcriptomic, and protein characterization. Our data supports that histological transdifferentiation from LUAD to LUSC or SCLC tumors is driven by epigenetic remodeling rather than by mutational events, and indicates that transdifferentiated tumors retain epigenomic features of their previous LUAD state. Integrative epigenomic, transcriptomic, and protein analysis revealed divergent biological pathways dysregulated for each histological outcome, such as upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses identified commonly dysregulated pathways in both SCLC- and LUSC-transdifferentiating tumors, including remarkable downregulation of a variety of immune-related pathways and upregulation of genes involved in the PRC2 epigenetic remodeling complex and AKT signaling. To validate drivers of transdifferentiation from our multi-omic data, preclinical in vivo experiments indicated that concurrent activation of AKT and MYC overexpression induced a LUSC phenotype with increased P40 and CK5/6 expression in EGFR -mutant LUAD patient-derived models. LUSC features in these models were further accentuated by the EGFR inhibitor osimertinib, which enriched for transdifferentiated LUSC cells. With the aim to validate potential therapeutic targets to prevent transdifferentiation from our multi-omic data, we tested the efficacy of EZH2 (the catalytic subunit of PRC2) or AKT pharmacological inhibitors in combination with osimertinib in an EGFR -mutant patient-derived xenograft model of LUAD-to-LUSC transdifferentiation and observed that inhibition of either pathway dramatically delayed relapse and prevented emergence of LUSC phenotypic markers. Additionally, pharmacological targeting of AKT and EGFR delayed relapse in a mixed LUAD/SCLC patient-derived xenograft model representing an intermediate step of LUAD-to-SCLC transdifferentiation. Interestingly, AKT inhibition selectively targeted the SCLC compartment of the tumor and prevented full SCLC transformation. This work was published in two manuscripts, of which I am the first and co-corresponding author (Quintanal-Villalonga et al., Cancer Discov 2021; Quintanal-Villalonga et al., J Hematol Oncol 2021) and defines a novel landscape of potential drivers and therapeutic vulnerabilities of histological transdifferentiation in lung cancer. Both transformed and de novo SCLCs are aggressive and rapidly metastatic lung tumors. Limited treatment options and transient responses translate to poor prognosis for patients with SCLC; 5-year survival rates are 200,000 annual deaths worldwide. Metastasis is the main cause of mortality among patients with SCLC. To characterize SCLC metastasis, we combined single-cell RNA sequencing (scRNA-seq) and multiplexed ion beam imaging (MIBI) technologies to study intratumoral heterogeneity and the surrounding tumor microenvironment (TME). Efforts to apply these technologies to human SCLC tumors have been limited, as surgical resections of primary tumors are performed in Citation Format: Alvaro Quintanal-Villalonga, Joseph M. Chan, Vianne R. Gao, Yubin Xie, Dana Pe’er, Charles M. Rudin. Multi-omic approaches to study the role of plasticity in therapy resistance and metastasis in 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 NG05.

Lineage plasticity, a capacity to reprogram cell phenotypic identity under evolutionary pressure, is implicated in treatment resistance and metastasis in multiple cancers. In lung adenocarcinomas (LUADs) amenable to treatment with targeted inhibitors, transformation to an aggressive neuroendocrine (NE) carcinoma resembling small cell lung cancer (SCLC) is a recognized mechanism of acquired resistance. Defining molecular mechanisms of NE transformation in lung cancer has been limited by a paucity of well annotated pre- and post-transformation clinical samples. We hypothesized that mixed histology LUAD/SCLC tumors may capture cancer cells proximal to, and on either side of, histologic transformation. We performed detailed genomic, epigenomic, transcriptomic and proteomic characterization of combined LUAD/SCLC tumors as well as pre- and post-transformation clinical samples. Our data support that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm in pre-transformation LUADs. Consistent shifts in gene expression programs in NE transformation include induction of several stem/progenitor cell regulatory pathways, including upregulation of PRC2 and WNT signaling, and suppression of Notch pathway activity. We observe induction of PI3K/AKT and an immunosuppressive phenotype in NE transformation. Taken together our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of NE transformation in lung cancer.

Lineage plasticity contributes to therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon drives neuroendocrine (NE) and squamous cell (LUSC) histologic transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is lower than that of either LUAD or de novo LUSC patients. To date, little is known about the molecular effectors enhancing lineage plasticity and driving histological transdifferentiation due to the paucity of well annotated pre- and post-transdifferentiation clinical samples amenable for molecular analyses. Currently no specific therapies for LUSC or NE transdifferentiation prevention are available for patients at high risk of transformation. We performed multi-omic profiling of transdifferentiating clinical samples, as well as control never-transformed LUAD and de novo LUSC and small cell carcinomas, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Our data suggest that histological transdifferentiation is driven by epigenetic -rather than mutational- events, and indicate that transdifferentiated tumors retain molecular features of their previous LUAD state. Integrative analysis revealed biological pathways dysregulated specifically for distinct histological outcomes, including downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses revealed commonly dysregulated pathways for transdifferentiated tumors, including marked downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in EGFR-mutant patient-derived xenograft transdifferentiation models. These results identify common and histology-specific drivers and dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to constrain lineage plasticity and prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Jacklynn Egger, Juan Qiu, Elisa de Stanchina, Natasha Rekhtman, Brian Houck-Loomis, Richard P. Koche, Helena A. Yu, Triparna Sen, Charles M. Rudin. AKT pathway as a therapeutic target to constrain lineage plasticity leading to histological transdifferentiation [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 658.

Small cell lung cancer (SCLC) is an aggressive disease characterized by early metastasis and exceptional lethality, comprising 13% of all lung cancer cases. With few treatment options, typically resulting in only transient responses, SCLC is responsible for approximately 250,000 deaths globally per year. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Here, we performed an in vitro CRISPR screen in SCLC cell lines from all major SCLC subtypes, including short-term cultured cells from patient-derived xenografts (PDXs), to identify potential therapeutic targets to enhance sensitivity to chemotherapy. Candidate hits were validated genetically and pharmacologically with in vitro synergy assays, in vivo clonal competition assays and pharmacologic assessments in PDX models. Signaling pathways were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Our CRISPR screen revealed the nuclear exporter exportin 1 (encoded by the XPO1 gene) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor or hematological malignancy, and demonstrated consistently high protein expression by IHC in clinical TMAs. A potent and selective exportin 1 inhibitor, selinexor, is approved for use in hematological malignancies. Combination of selinexor with cisplatin or irinotecan demonstrated synergy in vitro and efficacy in vivo in an array of chemonäive and chemoresistant SCLC PDXs, including all major SCLC subtypes. The combinations were well tolerated in mice. The chemo-sensitizing effects of selinexor were associated with suppression of chemotherapy-induced AKT activation. In conclusion, exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively, and these effects are independent of the SCLC subtype. These results provide preclinical rationale for the combination of selinexor with cisplatin or irinotecan in naïve and relapsed SCLC. The clinical availability of selinexor will allow rapid clinical translation of these results in a disease setting with extremely limited therapeutic options. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yuan Hao, Andrew Chow, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Juan Qiu, Elisa de Stanchina, Richard P. Koche, Triparna Sen, John T. Poirier, Charles M. Rudin. Exportin 1 inhibition as a therapeutic strategy for small cell lung cancer [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 3594.

e21166 Background: In lung adenocarcinomas (LUADs), lineage plasticity drives neuroendocrine (NE) and squamous cell (LUSC) transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is remarkably lower than those of LUAD or de novo LUSC patients. The paucity of transforming clinical specimens amenable for molecular analyses has hindered the identification of histological transformation drivers, and to date no specific therapies aimed to prevent or delay transdifferentiation-led therapy relapse are available for patients at high risk of transformation. Methods: We performed multi-omic profiling of LUAD-to-LUSC and LUAD-to-NE transdifferentiating clinical samples, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Clinical findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Results: Our data supports that histological transdifferentiation from LUAD to LUSC or NE tumors is driven by epigenetic remodeling rather than by mutational events, and indicate that transdifferentiated tumors retain epigenomic features of their previous LUAD state. Integrative epigenomic, transcriptomic and protein analysis revealed divergent biological pathways dysregulated for each histological outcome, such as downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses identified commonly dysregulated pathways in both NE- and LUSC-transdifferentiating tumors, including remarkable downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in different EGFR-mutant patient-derived xenograft transdifferentiation models. Conclusions: These results identify common and divergent dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation.

e20613 Background: Small cell lung cancer (SCLC) is an extremely aggressive disease comprising around 13% of lung cancer cases and responsible for approximately 250,000 deaths globally per year. A major cause of SCLC poor prognosis is the sparse treatment options available, typically resulting in only transient responses. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing only limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Methods: We performed an in vitro CRISPR screen in SCLC cell lines to identify potential therapeutic targets to sensitize to chemotherapy, including short-term cultured cell lines derived from patient-derived xenografts (PDXs). Candidate hits were validated genetically and pharmacologically with in vitro and in vivo. Signaling pathways involved in phenotypes observed were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Results: Our CRISPR screen revealed the nuclear exporter XPO1 (Exportin 1) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. Importantly, exportin 1 is a therapeutic target in hematological malignancies, counting with a potent and selective inhibitor, selinexor, approved for clinical use. Combination of selinexor with cisplatin or irinotecan demonstrated high synergy in vitro and exquisite efficacy i n vivo in an array of chemonäive and chemoresistant SCLC PDXs, respectively, including all major SCLC subtypes. These chemotherapy-sensitizing effects were associated with the ability of Exportin 1 to impair chemotherapy-induced AKT overactivation, potentially occurring as a cytoprotective/anti-apoptotic mechanism in response to chemotherapy. The combinations were well tolerated in mice. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor type or hematological malignancies, which we were able to confirm at the protein level in clinical TMAs. Conclusions: Exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively. These results provide preclinical rationale for the combination of selinexor with first line and second line chemotherapy in SCLC. The clinical availability of selinexor will allow immediate clinical translation of these results in a disease setting with extremely limited therapeutic options.

Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, accounting for about 15% of all lung cancer cases and approximately 200,000 deaths per year. SCLC genetic alterations are characterized by near-universal loss of function of the tumor suppressor genes TP53 and RB1. Several in-frame gene fusions have been identified in SCLC tumors and cell lines, with recurrent fusion events noted in up to 5% of cases. However, the biological and therapeutic implications of these fusions, including the recurrent in-frame RLF-MYCL1 gene fusion, are unknown. MYCL1 is frequently expressed in SCLC and differential expression of MYC family members has been associated with different SCLC subtypes. Here we investigate the functional significance of the RLF-MYCL1 gene fusion by developing a novel Rlf-Mycl1-driven genetically engineered mouse model (GEMM) of SCLC using CRISPR-Cas9 genome engineering. Methods: SgRNAs targeting intronic sequences of the Rlf and Mycl1 genes were cloned into a Cas9-expressing plasmid and co-transfected in NIH/3T3 cells. The presence of the Rlf-Mycl1 fusion was confirmed at the DNA and protein level. To test whether the RLF-MYCL1 gene fusion is important for the oncogenic switch during early SCLC development, we transduced Rb1Δ/Δ;Trp53Δ/Δ;Rbl2+/Δ precancerous neuroendocrine cells (preSCs), which were isolated from an early stage in tumorigenesis, with a lentiviral vector encoding Cas9 and with the dual sgRNAs targeting Rlf and Mycl1 genes (P-RM). Results: The P-RM transduced preSCs formed significantly more colonies in soft agar than those infected with non-targeting sgRNAs (P-NT) (p Summary: A major hurdle to SCLC clinical advancement is the absence of targetable driver mutations. Our findings indicate that the Rlf-Mycl1 gene fusion accelerates SCLC tumor growth. Defining mechanisms by which Rlf-Mycl1 accelerates tumorigenesis may identify therapeutic strategies to target SCLC harboring this fusion oncogene. Our study further supports a careful analysis of the involvement of other gene-fusions in SCLC. Citation Format: Metamia Ciampricotti, Triantafyllia Karakousi, Alvaro Quintanal Villalonga, Viola Allaj, Andrea Ventura, Triparna Sen, JT Poirier, Thales Papagiannakopoulos, Charles M. Rudin. Rlf-Mycl1 gene fusion as a novel oncogenic driver in a mouse model of small cell lung cancer [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 420.