Immunohistochemical profiling of small cell lung cancer reveals subtype heterogeneity and a tuft cell-like subtype characterized by LRMP and TRPM5.

9019 Background: The dominant expression of transcription factors ASCL1, NeuroD1, YAP1 or POU2F3 characteristically defines four small cell lung cancer (SCLC) subtypes (SCLC-A, SCLC-N, SCLC-Y and SCLC-P). The clinical validation and biological relevance of these emerging SCLC subtypes is currently lacking. Methods: Using the Illumina TruSeq RNA Exome Kit, we generated RNA-Seq data from 61 cases of SCLC and pulmonary carcinoid to interrogate gene expression differences in SCLC subtypes as well as in survival outliers (top and bottom decile) matched for clinically relevant prognostic factors and treatment. We also assessed YAP1 protein expression in a blinded fashion by immunohistochemistry in 130 SCLC cases. Results: We successfully classified 68% of SCLC into one of the four SCLC subtypes whereas 81.5% of carcinoids did not fit into any of these categories. GSEA for differentially expressed genes between outlier subgroups showed significant upregulation of interferon gamma and interferon alpha response genes in late survivors. Moreover, a previously validated 18-gene T-cell inflamed gene expression profile was upregulated in late survivors and in the SCLC-Y subtype. Furthermore, the SCLC-Y subtype and late survivors showed higher expression of HLA gene family and reduced expression of cancer testis antigens. The median (95%CI) OS was 14 (4.3, 28.8), 16.7 (0.9, NA), 8.1 (2, 9.7) and 20.1 (0.6, 39.5) months respectively, for SCLC-A, N, P and Y subtypes. YAP-1 protein expression was positive in 17 of 130 (13%) SCLC cases. The majority of cases with positive YAP1 expression by immunohistochemistry, 12 of 17 cases (70.6%), were limited stage SCLC at the time of original diagnosis. Conclusions: SCLC subtypes have clinical implication as predictive and prognostic biomarker. SCLC-Y subtype is enriched for T-cell inflamed phenotype and long term survival, and may predict for clinical benefit of immunotherapy.

The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial. To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue. Unexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype. SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC. See related commentary by Rekhtman, p. 1708.

<div>AbstractPurpose:<p>The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial.</p>Experimental Design:<p>To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue.</p>Results:<p>Unexpectedly, pathogenic mutations in <i>SMARCA4</i> were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile <i>SMARCA4</i>-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non–small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype.</p>Conclusions:<p>SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC.</p><p><i><a href="https://aacrjournals.org/clincancerres/article/doi/10.1158/1078-0432.CCR-24-0227" target="_blank">See related commentary by Rekhtman, p. 1708</a></i></p></div>

<div>AbstractPurpose:<p>The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial.</p>Experimental Design:<p>To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue.</p>Results:<p>Unexpectedly, pathogenic mutations in <i>SMARCA4</i> were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile <i>SMARCA4</i>-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non–small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype.</p>Conclusions:<p>SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC.</p><p><i><a href="https://aacrjournals.org/clincancerres/article/doi/10.1158/1078-0432.CCR-24-0227" target="_blank">See related commentary by Rekhtman, p. 1708</a></i></p></div>

Background - SCLC is the most aggressive lung cancer type and with the worst prognosis. There are four molecular subtypes based on the high expression of distinct transcription factors and with different therapeutic vulnerabilities. However, all share transcriptional addiction as pathogenic mechanism. Lurbinectedin is a novel oncogenic transcription inhibitor, approved in the United States and other countries for the treatment of adult patients with metastatic SCLC with disease progression on or after platinum-based chemotherapy. The aim of this study was to analyze the activity of lurbinectedin in the different SCLC molecular subtypes and to investigate new biomarkers of response. Methods and Results - We have characterized a panel of 20 SCLC human cell lines based on the expression of ASCL1, NEUROD1, YAP1 and POU2F3, where lurbinectedin yielded a mean IC50 value of 6.52 nM, which is considerable greater than the activity exerted by topotecan, irinotecan, carboplatin, etoposide, olaparib, alisertib and navitoclax (IC50 100 µM-100 nM). Lurbinectedin potency is high in the four molecular subtypes, with IC50 values of 4.1, 14.9, 7.2, and 0.2 nM for SCLC-A, -N, -I and -P, respectively. However, high expression of POU2F3 correlated with better responses. In fact, mean IC50 was 0.28 nM for POU2F3high cells versus 12.1 nM for all POU2F3low cells (p=0.0443). Additionally, basal SLFN11 levels were evaluated, observing that SLFN11high cells responded better to lurbinectedin, with IC50 values of 1.1 nM versus 11.8 nM for SLFN11low (p=0.05). Likewise, lurbinectedin treatment induced greater antitumor activity in SLFN11high tumor-bearing mice (H526: T/C, 18% on day 11) than in SLFN11low tumor-bearing mice (H82: T/C, 65% on day 7) after intravenous treatment at 0.18 mg/kg (on days 0, 7 and 14). Finally, SLFN11 expression was evaluated by IHC in FFPE tumor samples from SCLC patients participating in a multicenter phase II clinical trial in advanced solid tumors (NCT02454972), which allowed lurbinectedin accelerated approval in this indication by FDA. Patients with higher expression of SLFN11 had a slightly better overall survival (OS at 6 months: 68.4% (<15%, N=20) vs. 98.7% (≥15%, N=20) p=0.0261)), especially in the refractory/resistant subgroup (OS at 6 months: 33.3% (<15%, N=9) vs. 100.0% (≥15%, N=6) p<0.0001). Conclusions - Lurbinectedin is highly effective in all molecular subtypes of SCLC in vitro and in vivo, with IC50 values at least two logs more potent than for other antitumoral agents and its activity is even greater in tumors with high POU2F3 expression and/or high SLFN11 expression. Citation Format: Marta Martínez Diez, Gema Santamaría Nuñez, María José Guillén, Daniel Rueda, Eva Maria Garrido-Martin, Pablo Avilés, Carmen Cuevas. Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response. [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 6247.

<div>Abstract<p>Genomic studies support the classification of small cell lung cancer (SCLC) into subtypes based on the expression of lineage-defining transcription factors ASCL1 and NEUROD1, which together are expressed in ∼86% of SCLC. ASCL1 and NEUROD1 activate SCLC oncogene expression, drive distinct transcriptional programs, and maintain the <i>in vitro</i> growth and oncogenic properties of ASCL1 or NEUROD1-expressing SCLC. ASCL1 is also required for tumor formation in SCLC mouse models. A strategy to inhibit the activity of these oncogenic drivers may therefore provide both a targeted therapy for the predominant SCLC subtypes and a tool to investigate the underlying lineage plasticity of established SCLC tumors. However, there are no known agents that inhibit ASCL1 or NEUROD1 function. In this study, we identify a novel strategy to pharmacologically target ASCL1 and NEUROD1 activity in SCLC by exploiting the nuclear localization required for the function of these transcription factors. Karyopherin β1 (KPNB1) was identified as a nuclear import receptor for both ASCL1 and NEUROD1 in SCLC, and inhibition of KPNB1 led to impaired ASCL1 and NEUROD1 nuclear accumulation and transcriptional activity. Pharmacologic targeting of KPNB1 preferentially disrupted the growth of ASCL1<sup>+</sup> and NEUROD1<sup>+</sup> SCLC cells <i>in vitro</i> and suppressed ASCL1<sup>+</sup> tumor growth <i>in vivo</i>, an effect mediated by a combination of impaired ASCL1 downstream target expression, cell-cycle activity, and proteostasis. These findings broaden the support for targeting nuclear transport as an anticancer therapeutic strategy and have implications for targeting lineage-transcription factors in tumors beyond SCLC.</p>Significance:<p>The identification of KPNB1 as a nuclear import receptor for lineage-defining transcription factors in SCLC reveals a viable therapeutic strategy for cancer treatment.</p></div>

<div>Abstract<p>Genomic studies support the classification of small cell lung cancer (SCLC) into subtypes based on the expression of lineage-defining transcription factors ASCL1 and NEUROD1, which together are expressed in ∼86% of SCLC. ASCL1 and NEUROD1 activate SCLC oncogene expression, drive distinct transcriptional programs, and maintain the <i>in vitro</i> growth and oncogenic properties of ASCL1 or NEUROD1-expressing SCLC. ASCL1 is also required for tumor formation in SCLC mouse models. A strategy to inhibit the activity of these oncogenic drivers may therefore provide both a targeted therapy for the predominant SCLC subtypes and a tool to investigate the underlying lineage plasticity of established SCLC tumors. However, there are no known agents that inhibit ASCL1 or NEUROD1 function. In this study, we identify a novel strategy to pharmacologically target ASCL1 and NEUROD1 activity in SCLC by exploiting the nuclear localization required for the function of these transcription factors. Karyopherin β1 (KPNB1) was identified as a nuclear import receptor for both ASCL1 and NEUROD1 in SCLC, and inhibition of KPNB1 led to impaired ASCL1 and NEUROD1 nuclear accumulation and transcriptional activity. Pharmacologic targeting of KPNB1 preferentially disrupted the growth of ASCL1<sup>+</sup> and NEUROD1<sup>+</sup> SCLC cells <i>in vitro</i> and suppressed ASCL1<sup>+</sup> tumor growth <i>in vivo</i>, an effect mediated by a combination of impaired ASCL1 downstream target expression, cell-cycle activity, and proteostasis. These findings broaden the support for targeting nuclear transport as an anticancer therapeutic strategy and have implications for targeting lineage-transcription factors in tumors beyond SCLC.</p>Significance:<p>The identification of KPNB1 as a nuclear import receptor for lineage-defining transcription factors in SCLC reveals a viable therapeutic strategy for cancer treatment.</p></div>

The identification of KPNB1 as a nuclear import receptor for lineage-defining transcription factors in SCLC reveals a viable therapeutic strategy for cancer treatment.

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<0.001, FC=81), high frequency of CDKN2A homozygous deletion and SMARCA4 mutations, as well as intact RB1. These features are distinct from SCLC, wherein transcriptional subtypes lack distinct genomic characteristics. Consistent with CDKN2A deletion, YAP1-high pLCNEC cell lines have increased sensitivity to MEK1 and CDK4/6 inhibition. We also demonstrate that RB1 loss downregulates YAP1 expression, which may account for the absence of YAP1 in treatment-naïve SCLC due to ubiquitous loss of RB1. In contrast to treatment-naïve SCLC, where our group and others have been unable to detect YAP1, single-cell RNAseq analysis of biopsies from patients with relapsed SCLC identified emerging YAP1-positive cancer cell populations, which are similarly associated with increased EMT, immune cell infiltration (CD8+ T-cells), and loss of NE gene expression. This suggests that the ability for cancer cells to acquire YAP1 expression and, perhaps, pLCNEC-like features, may be a resistance mechanism in relapsed SCLC, contributing to the abundant intratumoral heterogeneity and highlighting potential vulnerabilities to overcome resistance. In summary, YAP1 may be a predictive biomarker of intact RB1 and response to cellular and checkpoint immunotherapy and MEK1/CDK4/6 inhibition in pLCNEC and relapsed SCLC. 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.

SCLC-CellMiner: A Resource for Small Cell Lung Cancer Cell Line Genomics and Pharmacology Based on Genomic Signatures

Small cell lung cancer (SCLC) is a deadly, high grade neuroendocrine (NE) tumor without recognized morphologic heterogeneity. However, over 30 years ago we described a SCLC subtype with "variant" morphology which did not express some NE markers and exhibited more aggressive growth. To quantitate NE properties of SCLCs, we developed a 50-gene expression-based NE score that could be applied to human SCLC tumors and cell lines, and genetically engineered mouse (GEM) models. We identified high and low NE subtypes of SCLC in all of our sample types, and characterized their properties. We found that 16% of human SCLC tumors and 10% of SCLC cell lines were of the low NE subtype, as well as cell lines from the GEM model. High NE SCLC lines grew as non-adherent floating aggregates or spheroids while Low NE lines had morphologic features of the variant subtype and grew as loosely attached cells. While the high NE subtype expressed one of the NE lineage master transcription factors ASCL1 or NEUROD1, together with NKX2-1, the entire range of NE markers, and lacked expression of the neuronal and NE repressor REST, the low NE subtype had lost expression of most NE markers, ASCL1, NEUROD1 and NKX2-1 and expressed REST. The low NE subtype had undergone epithelial mesenchymal transition (EMT) and had activated the Notch, Hippo and TGFβ pathways and MYC oncogene . Importantly, the high and low NE group of SCLC lines had similar gene expression profiles as their SCLC tumor counterparts. SCLC tumors and cell lines can exhibit distinct inter-tumor heterogeneity with respect to expression of NE features. Loss of NE expression results in major alterations in morphology, growth characteristics, and molecular properties. These findings have major clinical implications as the two subtypes are predicted to have very different responses to targeted therapies.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

Background: Small cell lung cancer (SCLC) is a highly lethal malignancy, with rapidly acquired chemotherapy resistance. Some studies have reported that Wnt signaling pathway activation promoted cell proliferation and was correlated with chemo-resistance in SCLC. None of the therapies targeting Wnt pathway components of the transmembrane and the cytoplasm have been successful in a clinical application due to toxicity and insufficient efficacy. However, targeting Wnt signaling inside the nucleus has been drawing increasing attention as cancer therapeutics. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers for cell proliferation, thus TNIK is expected as a novel druggable target. On the other hand, the question remains whether TNIK is a critical target in SCLC. We hypothesize that a TNIK inhibitor has potent anti-tumor effects in SCLC and its promising biomarkers exist. Methods: We used 29 SCLC cell lines including all four subtypes defined by differential expression of transcription factors ASCL1, NEUROD1, POU2F3, and inflamed gene signature (SCLC-A, N, P, and I, respectively) to evaluate the effect of a TNIK inhibitor, NCB-0846 in vitro. We correlated NCB-0846 IC50 values with proteomic profiling (Reverse Phase Protein Array, RPPA) data. Protein expression was examined by western blotting. Results: NCB-0846 markedly reduced cell proliferation in SCLC-N and P cell lines. There was a strong positive correlation between cMyc and the efficacy of NCB-0846 (r=-0.484, P<0.01), while a negative correlation between TTF1 and the efficacy (r=0.569, P<0.01). Additionally, NCB0846 decreased the expression of cMyc in cMyc-high/TTF-1 low SCLC cells. Conclusions: These findings indicate that TNIK inhibitors may be a new personalized molecular-targeted therapy in cMyc-high/TTF-1-low SCLC. Citation Format: Azusa Tanimoto, Robert J. Cardnell, Benjamin B. Morris, Kavya Ramkumar, Shen Li, Qi Wang, Allison C. Stewart, Carl Michael Gay, Jing Wang, Lauren Averett Byers. TNIK inhibition as a novel therapeutic in cMyc high/TTF1 low SCLC. [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 3854.

Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities.

MYC Drives Temporal Evolution of Small Cell Lung Cancer Subtypes by Reprogramming Neuroendocrine Fate